Este trabalho foi financiado pelo NIH concede AI56168, AI71142, AI87541 e AI100631 de MDP. Maurizio Del Poeta é Burroughs Wellcome Investigator em Doenças Infecciosas.

1. Colesterol depleção de células J774A.1 com MβCD <ol><li> Em uma cabine de segurança biológica estéril, sementes células tipo macrófagos J774A.1 5 10 por poço numa placa de 96 poços de cultura de células em 200 ul de Dulbecco Modified Eagle Médium (DMEM) suplementado com 10% de soro fetal bovino (FBS) e 1% penicilina / estreptomicina (P / S). Incubar a 37 ° C e 5% de CO 2 O / N. </li><li> Remover mídia da monocamada de células e lava-se as células duas vezes com solução salina tamponada com fosfato 1x (PBS) que foi filtrado e autoclavado. </li><li> Adicionar 200 ul de solução MβCD na concentração desejada (10 mM mM ou 30 em PBS) ou 1x PBS como um controlo e incubar durante 30 min a 37 ° C com agitação. Remover o sobrenadante e reserva a RT para a análise quantitativa com um kit disponível comercialmente imediatamente após o procedimento. </li><li> Lavar as células duas a três vezes com PBS 1x ou DMEM isento de soro e continuar com infecção ou lisam as células por pipetting de duas a três vezes com H2O desionizada para análise com cromatografia de camada fina ou com um kit. NOTA: Após a depleção de colesterol um kit de colesterol quantificação disponíveis comercialmente podem ser utilizados. Consulte a seção de materiais para obter detalhes. Siga as instruções do fabricante, conforme escrito. </li></ol> 2. Observação de Colesterol conteúdo por cromatografia em camada delgada (TLC) <ol><li> Lavar um tanque TLC duas vezes com acetona e uma vez com uma solução de éter de petróleo: éter dietílico: ácido acético (65: 30: 1 por volume). Saturar o tanque com o éter de petróleo: éter dietílico: ácido acético (65: 30: 1 por volume) e solução deixar S / N. NOTA: Organic solventes deve sempre ser usado sob uma coifa para evitar a inalação de vapor. Luvas e jaleco deve ser usado em todos os momentos. O ácido acético é um ácido forte e devem ser utilizados com cautela. </li><li> Em um gabinete de biossegurança estéril, semente 10 macrófagos-like 6 J774A.1 por poço em uma de 6 poços cell placa de cultura num volume de 5 ml de DMEM morno, suplementado com 10% FBS e 1% de P / S. Incubar a 37 ° C, 5% de CO 2 O / N. </li><li> Esgotar macrófagos de colesterol, seguindo passos 1,2-1,4, substituindo 1 ml para 200 ul onde aplicável para explicar o maior tamanho bem. </li><li> Adicionar 500 ul de Tripsina-EDTA a cada poço, incubar durante 3 min a 37 ° C, e raspe as células com um raspador de células. </li><li> Transferir para um tubo de microcentrífuga e adicionar um adicional de 500 ul de DMEM morno, suplementado com 10% FBS e 1% de P / S. </li><li> Rodar as células durante 5 min a 300 xg e remover o sobrenadante. </li><li> Adicionar um adicional de 500 ul de DMEM morno, suplementado com 10% FBS e 1% de P / S para a pelete de células e ressuspender cuidadosamente, pipetando para cima e para baixo. </li><li> Retirar 10 ml de células e contagem de células em um hemocitômetro. Normalizar as concentrações de células a partir de cada amostra e adicionar um número igual de células para tubos de vidro. NOTA: Neste passo, um maioescolher a combinação de células a partir dos mesmos grupos de tratamento para se obter um extracto mais concentrado final de lípido. </li><li> Centrifugar células a 300 xg por 5 min a RT e remover mídia. </li><li> Adicionar 2 ml de metanol e vortex. Em seguida, adicione 1 ml de clorofórmio e vortex. Verifique o estado de fase para garantir que a solução em monofásico. NOTA: Os tubos podem ser armazenados a 4 ° CO / N. </li><li> Centrifugar a 1.700 xg durante 10 min à temperatura ambiente e transferir o sobrenadante para um novo tubo </li><li> Adicionar um adicional de 1 ml de clorofórmio, seguido de 1 ml de dH 2 O. Vortex duas vezes durante 30 segundos. Centrifugar a 1.700 xg durante 5 min à temperatura ambiente. </li><li> Pesar um tubo de vidro sobre uma balança sensível e utilizar uma pipeta de Pasteur de vidro para transferir a fase mais baixa no tubo de vidro de peso conhecido. </li><li> Seque-se lipídios em um evaporador centrífugo até secar (cerca de 2 horas). Pesar tubo com lípidos secos e calcular o peso seco de lípidos. NOTA: lípidos secos podem ser armazenados em -20 ° C até estar pronto para executar TLC. </li><li> Dilui-se lípidos secos em clorofórmio suficiente para normalizar a concentração de lípido (geralmente 20 - 50 ul) e 20 ul de carga do lípido diluído em uma placa de TLC de sílica. Carga de 20 ug de colesterol diluída em 20 ul de clorofórmio como um padrão. </li><li> Adicionar placa TLC seco para o tanque TLC saturado e permitir solvente migrar até 1 cm antes da borda da placa. Retire a placa de TLC de tanque e deixe-a secar cerca de 5 min. </li><li> Visualize lipídios, colocando em um tanque de vapor de iodo para verificar a migração. Remover manchas e permitir a desvanecer-se para cerca de 10 - 15 min sob o capô. NOTA: O iodo é um perigo de inalação. Utilize sempre sob uma coifa. </li><li> Prepara-se uma solução para a coloração de lípidos neutros através da combinação de 60 ml de metanol com 60 ml de H2O desionizada, 4 ml de ácido sulfúrico, e 630 mg de cloreto de manganês. </li><li> Cuidadosa e lentamente mergulhar na placa de TLC em solução de coloração de lípidos neutros em uma bandeja e remover sem desprendendo-se a sílica layer. NOTA: A solução de coloração lípido neutro pode ser reutilizado várias vezes e por isso pode ser recuperado a partir da bandeja e colocados em um frasco de volta para uso posterior. </li><li> Permitir que a placa secar sob o capô à temperatura ambiente até que todas as bolhas desapareceu. Aquecer a placa de TLC em um conjunto de blocos de calor a 160 ° C e char para a cor desejada. NOTA: Um programa de densitometria como funciona a visão LS pode ser utilizado para quantificar as bandas carbonizados para comparar amostras de lipídios. </li></ol> 3. A infecção de macrófagos com C. neoformans (H99) <ol><li> Em uma cabine de segurança biológica estéril, semente 10 5 células semelhantes a macrófagos por cavidade em uma placa de 96 poços de cultura de células em 200 ul de DMEM suplementado com 10% FBS e 1% de P / S. Incubar a 37 ° C, 5% CO 2, 5% de CO 2 O / N. NOTA: A infecção também pode ser feito de vidro de fundo pratos confocal para imagens mais fácil; todas as quantidades permanecem as mesmas. </li><li> Crescer uma cultura de C. neoformans (H99)através da inoculação de 10 ml de YNB com uma colónia obtida a partir de uma placa atingido e incubando-O / N a 30 ° C com agitação. </li><li> Lave e contar C. neoformans (H99) células. <ol><li> Centrífuga C. neoformans cultura O / N a 1700 xg durante 10 min a 4 ° C. </li><li> Remova a mídia e descarte. Lavar as células com 5 ml de PBS 1x. Centrifugar a 1.700 xg durante 10 min a 4 ° C. </li><li> Remover PBS e lavar com 5 ml de PBS 1x filtrada. Centrifugar a 1.700 xg durante 10 min a 4 ° C. Repita este passo mais 2 vezes. </li><li> Remover PBS e ressuspender em 5 ml de PBS 1x. </li><li> Fazer uma diluição em série em PBS para obter uma diluição de 1: 500 a cultura lavada. <ol><li> Adicionar 100 ul da amostra original de 900 ul de 1x PBS para obter uma diluição de 1:10. </li><li> Adicionar 100 ul de amostra diluída a 1:10 900 ul de 1x PBS para obter uma diluição de 1: 100. </li><li> Adicionar 200 ul de 1: 100 da amostra diluída para 800 ul de 1x PBS para se obter uma mistura 1: 500 dilutiem </li></ol></li><li> Tomar 10 ul de 1: 500 de diluição e contar com hemocitómetro para calcular o número de células. </li></ol></li><li> Prepare a solução de trabalho para ativar macrófagos e opsonizantes C. neoformans. <ol><li> Diluir LPS e IFN-y 100x de soluções estoque, adicionando 10 ml para 990 mL. NOTA: LPS e IFN-y são utilizados para aumentar a absorção fagocíticas, mas não são necessárias para a fagocitose. Se houver um interesse na actividade fungicida de macrófagos, realizar activação de O / N a 37 ° C com agitação antes da infecção. </li><li> Por exemplo combinar 7,5 ul de LPS diluídas, 1,25 ul de IFNy diluída, 1,25 ul de anticorpo GXM e o volume da C. cultura neoformans que dá 1,25 x 10 5 células. Levar o volume até 250 ul multiplicado pelo número de amostras com DMEM suplementado com 10% FBS e 1% de P / S. </li><li> Vortex e incubar solução durante 20 min a 37 ° C com agitação. NOTA:Depleção de colesterol (passos 1,2-1,4) pode ser feita simultaneamente com o passo de opsonização. Certifique-se de tratar os macrófagos antes de se combinar a solução de trabalho, como as células opsonizadas deve ser otimamente não é mais usado do que 20 minutos após o passo 3.4.3 incubação. </li></ol></li><li> Infectam macrófagos <ol><li> Macrófagos lavar duas vezes com DMEM isento de soro e adicionar 200 mL de opsonizado C. neoformans solução de trabalho a cada poço. </li><li> Incubar durante 2 horas a 37 ° C. </li></ol></li><li> Fixar e corar as células <ol><li> Remova a mídia e lavar as células duas vezes com DMEM. </li><li> Secar a monocamada de células durante 10 min e adicionar 200 uL de metanol arrefecido em gelo para fixar as células. </li><li> Incubar durante 15 minutos à temperatura ambiente e remover qualquer metanol remanescente. </li><li> Adicionar 200 ul de 10x Giemsa e incubar durante 5 min à TA. </li><li> Lave 2-3 vezes com água deionizada e seco O / N com tampa off. NOTA: Imagem pode ser feito no dia seguinte ou até uma semana followicoloração ng. </li></ol></li><li> Visualize e Contagem <ol><li> Usando um microscópio, a contagem de 300 células por ponto de dados (se houver 2 do mesmo tratamento contar 150 por poço) e anote o número de macrófagos infectados e número de células Cryptococcus engoliram. NOTA: Para garantir ainda amostragem per use ponto de dados 2 placas por estado, escolher 3 áreas não sobrepostas por placa e conte 50 células por área. </li><li> Calcular o índice fagocitário multiplicando a percentagem de macrófagos infectados pelo número significativo de C. neoformans por macrófago. Normalizar o índice fagocitário, expressando os valores como uma percentagem do controlo tratado com PBS 1x. Depois de vários ensaios calcular o valor médio e o desvio padrão da média para determinar as tendências no índice de fagocitose. Use t de Student para determinar a significância. </li><li> Tome micrografias das células de 1000X ou aumento de 400X. </li></ol></li></ol> 4. Ensaio de Azul de Tripano <ol><li> Em uma cabine de segurança biológica estéril, semente 10 6 células tipo macrófagos por cavidade em uma placa de 6 poços de cultura de células num volume de 5 ml de meio essencial mínimo de Dulbecco (DMEM) suplementado com 10% de soro fetal bovino e 1% de penicilina / estreptomicina . Incubar a 37 ° C, 5% de CO 2 O / N. </li><li> Esgotar macrófagos de colesterol, seguindo passos 1,2-1,4 substituindo 2 ml para 200 ul onde aplicável para explicar o maior tamanho bem. NOTA: Certifique-se de incluir um controle tratado com 1x PBS, bem como um controle que é raspado antes de qualquer tratamento. </li><li> Adicionar 500 ul de 1x PBS a cada poço e suavemente raspar as células com um raspador de células. Transferir para um tubo de microcentrífuga e suspender as células cuidado pipetando para cima e para baixo. </li><li> Retirar 10 ml de células e mancha com 1 ml de 4% Trypan azul. </li><li> Contagem de células em um hemocitômetro e calcular a viabilidade usando a seguinte equação:% de viabilidade = [1 - (células azuis / células totais)]x 100. Os valores de normalização para o controle que não foi tratado. Depois de vários ensaios calcular o valor médio eo desvio-padrão para determinar as tendências da viabilidade. Use t de Student para determinar a significância. </li></ol>

<ol>
	<li>Sarantis, H., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subversion+of+phagocytosis+for+pathogen+survival.">Subversion of phagocytosis for pathogen survival.</a> Cell Host &amp; Microbe. 12 (4), 419-431 (2012).</li><li>Rougerie, P., Miskolci, V., Cox, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+Of+Membrane+Structures+During+Phagocytosis+And+Chemotaxis+Of+Macrophages:+Role+And+Regulation+Of+The+Actin+Cytoskeleton.">Generation Of Membrane Structures During Phagocytosis And Chemotaxis Of Macrophages: Role And Regulation Of The Actin Cytoskeleton.</a> Immunological Reviews. 256 (1), 222-239 (1111).</li><li>Liu, T., Perlin, D. S., Xue, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+Of+Cryptococcal+Meningitis.">Molecular Mechanisms Of Cryptococcal Meningitis.</a> Virulence. 3, 173 (2012).</li><li>Abadi, J., Pirofski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Antibodies+Reactive+With+The+Cryptococcal+Capsular+Polysaccharide+Glucuronoxylomannan+Are+Present+In+Sera+From+Children+With+And+Without+Human+Immunodeficiency+Virus+Infection.">A Antibodies Reactive With The Cryptococcal Capsular Polysaccharide Glucuronoxylomannan Are Present In Sera From Children With And Without Human Immunodeficiency Virus Infection.</a> The Journal Of Infectious Diseases. 180 (3), 915-919 (1999).</li><li>Kechichian, T. B., Shea, J., Del Poeta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+Of+Alveolar+Macrophages+Decreases+The+Dissemination+Of+A+Glucosylceramide-Deficient+Mutant+Of+Cryptococcus+Neoformans+In+Immunodeficient+Mice.">Depletion Of Alveolar Macrophages Decreases The Dissemination Of A Glucosylceramide-Deficient Mutant Of Cryptococcus Neoformans In Immunodeficient Mice.</a> Infection And Immunity. 75 (10), 4792-4798 (2007).</li><li>Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococci+At+The+Brain+Gate:+Break+And+Enter+Or+Use+A+Trojan+Horse.">Cryptococci At The Brain Gate: Break And Enter Or Use A Trojan Horse.</a> The Journal Of Clinical Investigation. 120 (5), 1389-1692 (1172).</li><li>Chr&#233;tien, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+Of+Cerebral+Cryptococcus+Neoformans+Infection+After+Fungemia.">Pathogenesis Of Cerebral Cryptococcus Neoformans Infection After Fungemia.</a> The Journal Of Infectious Diseases. 186 (4), 522-530 (2002).</li><li>Luberto, C., Martinez-Mari&#241;o, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+Of+App1+As+A+Regulator+Of+Phagocytosis+And+Virulence+Of+Cryptococcus+Neoformans.">Identification Of App1 As A Regulator Of Phagocytosis And Virulence Of Cryptococcus Neoformans.</a> The Journal Of Clinical Investigation. 112 (7), 1080-1094 (1172).</li><li>Mcquiston, T. J., Williamson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradoxical+Roles+Of+Alveolar+Macrophages+In+The+Host+Response+To+Cryptococcus+Neoformans.">Paradoxical Roles Of Alveolar Macrophages In The Host Response To Cryptococcus Neoformans.</a> Journal Of Infection And Chemotherapy: Official Journal Of The Japan Society Of Chemotherapy. 18 (1), 1-9 (2012).</li><li>Garc&#237;a-Rodas, R., Zaragoza, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catch+Me+If+You+Can:+Phagocytosis+And+Killing+Avoidance+By+Cryptococcus+Neoformans.">Catch Me If You Can: Phagocytosis And Killing Avoidance By Cryptococcus Neoformans.</a> FEMS Immunology And Medical Microbiology. 64 (2), 147-161 (2012).</li><li>Coelho, C., Bocca, A. L., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Intracellular+Life+Of+Cryptococcus+Neoformans.">The Intracellular Life Of Cryptococcus Neoformans.</a> Annual Review Of Pathology. 9. 219, 10-1146 (2014).</li><li>Rao, M., Peachman, K. K., Alving, C. R., Rothwell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+Of+Cellular+Cholesterol+Interferes+With+Intracellular+Trafficking+Of+Liposome-Encapsulated+Ovalbumin.">Depletion Of Cellular Cholesterol Interferes With Intracellular Trafficking Of Liposome-Encapsulated Ovalbumin.</a> Immunology And Cell Biology. 81, 415-423 (2003).</li><li>Sein, K. K., Aikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Prime+Role+Of+Plasma+Membrane+Cholesterol.">The Prime Role Of Plasma Membrane Cholesterol.</a> In The Pathogenesis Of Immune Evasion And Clinical Manifestations Of Falciparum Malaria. Medical Hypotheses. 51 (2), 10-1016 (1998).</li><li>Pucadyil, T. J., Tewary, P., Madhubala, R., Chattopadhyay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+Is+Required+For+Leishmania+Donovani+Infection:+Implications+In+Leishmaniasis.">Cholesterol Is Required For Leishmania Donovani Infection: Implications In Leishmaniasis.</a> Molecular And Biochemical Parasitology. 133, 145-152 (2004).</li><li>Tagliari, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Microdomain+Components+Of+Histoplasma+Capsulatum+Yeast+Forms,+And+Their+Role+In.">Membrane Microdomain Components Of Histoplasma Capsulatum Yeast Forms, And Their Role In.</a> Alveolar Macrophage Infectivity. Biochimica Et Biophysica Acta. 1818 (3), 458-466 (2012).</li><li>Crane, J. M., Tamm, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+Of+Cholesterol+In+The+Formation+And+Nature+Of+Lipid+Rafts+In+Planar+And+Spherical+Model+Membranes.">Role Of Cholesterol In The Formation And Nature Of Lipid Rafts In Planar And Spherical Model Membranes.</a> Biophysical Journal. 86 (5), 2965-2979 (2004).</li><li>Brown, D. A., London, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+Of+Lipid+Rafts+In+Biological+Membranes.">Functions Of Lipid Rafts In Biological Membranes.</a> Annual Review Of Cell And Developmental Biology. 14, 111-136 (1998).</li><li>Simons, K., Toomre, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Rafts+And+Signal+Transduction+Nature+Reviews.">Lipid Rafts And Signal Transduction Nature Reviews.</a> Molecular Cell Biology. 1 (1), 31-39 (2000).</li><li>Ilangumaran, S., Hoessli, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+Of+Cholesterol+Depletion+By+Cyclodextrin+On+The+Sphingolipid+Microdomains+Of+The+Plasma+Membrane.">Effects Of Cholesterol Depletion By Cyclodextrin On The Sphingolipid Microdomains Of The Plasma Membrane.</a> The Biochemical Journal. 335 (Pt 2), 433-440 (1998).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+Method+Of+Total+Lipid+Extraction+And+Purification).">A Rapid Method Of Total Lipid Extraction And Purification).</a> Canadian Journal Of Biochemistry And Physiology. 37 (8), 911-917 (1959).</li><li>Mukherjee, S., Lee, S., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+To+Cryptococcus+Neoformans+Glucuronoxylomannan+Enhance+Antifungal+Activity+Of+Murine+Macrophages.">Antibodies To Cryptococcus Neoformans Glucuronoxylomannan Enhance Antifungal Activity Of Murine Macrophages.</a> Infect. Immun. 63 (2), 573-579 (1995).</li><li>Deshaw, M., Pirofski, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+To+The+Cryptococcus+Neoformans+Capsular+Glucuronoxylomannan+Are+Ubiquitous.">Antibodies To The Cryptococcus Neoformans Capsular Glucuronoxylomannan Are Ubiquitous.</a> In Serum From HIV+ And HIV- Individuals. Clinical And Experimental Immunology. 99 (3), 425-432 (1995).</li><li>Tripathi, K., Mor, V., Bairwa, N. K., Del Poeta, M., Mohanty, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyurea+Treatment+Inhibits+Proliferation+Of+Cryptococcus+Neoformans+In+Mice.">Hydroxyurea Treatment Inhibits Proliferation Of Cryptococcus Neoformans In Mice.</a> Frontiers In Microbiology. 3, 187 (2012).</li><li>Chaka, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+Of+Phagocytosis+And+Killing+Of+Cryptococcus+Neoformans+By+Human+Peripheral+Blood+Mononuclear+Cells+By+Flow+Cytometry.">Quantitative Analysis Of Phagocytosis And Killing Of Cryptococcus Neoformans By Human Peripheral Blood Mononuclear Cells By Flow Cytometry.</a> Clin. Diagn. Lab. Immunol. 2 (6), 753-759 (1995).</li></ol>

The authors have nothing to disclose.

Ao trabalhar com este protocolo é importante para obter a contagem de células precisos ao cultivo de células de mamíferos e opsonizantes C. células neoformans. Isto minimiza a variação entre ensaios e garante uma precisas 1: 1 de alvo da razão efector ao longo do estudo. Também é crítico para coordenar o momento da depleção de colesterol e para prevenir a infecção das células de levedura ou células opsonizadas macrófagos tratados de repouso à temperatura ambiente entre os processos. ...

A fagocitose é um processo pelo qual as entidades extracelulares são internalizados pelas células hospedeiras. É uma arma fundamental no arsenal do sistema imunológico para se defender contra patógenos, mas o processo pode muitas vezes ser subvertida por patógenos para permitir a internalização e se espalhando por todo o corpo 1. A fagocitose é mediada por vários eventos de sinalização que resultam em anexo e imersão através de rearranjos do citoesqueleto da célula hospedeira. Fagócitos &quot;profissionais&quot; são capazes de reconhecer e ligar-se a opsoninas sobre a superfície do agente patogénico invasor para sinalizar para a ligação e a formação de lamelipï¿½ios, que engolir o patógeno e formar um fagossoma 2. Entre as chamadas fagócitos &#39;profissionais&#39; são os macrófagos. Os macrófagos são células altamente especializadas que realizam funções de proteção que incluem procurando e eliminando os agentes que causam doenças, reparar tecidos danificados, e mediação da inflamação, a maioria dosestes através do processo de fagocitose 1,2. Cryptococcus neoformans é uma espécie de fungo patogênico que causa uma doença grave conhecida como criptococose. Esporos Cryptococcus são inaladas pelo acolhimento e resultar em uma infecção pulmonar, que é geralmente assintomática. Pensa-se que a exposição é extremamente predominante; uma amostra de 61 crianças a partir dos Infectologia Pediátrica do Hospital Clinic Center Bronx-Lebanon descobriu que todos os entrevistados tinham anticorpos para o glucuronoxylomannan polissacarídeo cryptococcal e outros estudos têm mostrado prevalência em ambos os vírus da imunodeficiência humana (HIV) não infectado e adultos infectados 3, 4. macrófagos alveolares são a primeira linha de resposta à infecção pulmonar e na maioria dos casos com sucesso claro o patógeno. No entanto, em indivíduos imunodeprimidos (pacientes por exemplo, HIV e AIDS) o fermento é capaz de sobreviver dentro dos macrófagos. Nestescasos, os macrófagos podem servir como um nicho para a replicação do patógeno e pode facilitar a sua difusão para o sistema nervoso central (SNC) em que a doença se torna fatal 5-8. Pensa-se que os macrófagos podem até mesmo entregar o fermento diretamente nas meninges, ajudando a levedura de atravessar a barreira hemato-encefálica através do modelo de &quot;cavalo de Tróia&quot; 3,9 - 11. Assim, é importante compreender o processo de fagocitose e os factores que a afectam, especialmente na infecção pelo fungo. Trabalhos anteriores em outros sistemas de patógenos apontam para colesterol e lipídios jangadas formadas por colesterol como tendo um papel importante a desempenhar na fagocitose 12-15. O colesterol é as espécies mais abundantes de lípidos nas células de mamíferos e compreende 25 - 50% da membrana da célula de mamífero 16. Verificou-se que desempenham um papel na modulação da BIOPpropriedades hysical de membranas, alterando a sua rigidez 17. O colesterol e esfingolípidos em conjunto formam microdomínios lipídicos dentro da membrana conhecida como jangadas lipídicas. Jangadas lipídicas foram encontrados para ser envolvida na formação de caveolas, bem como o fornecimento de um domínio isolado para certos tipos de sinalização 16-18. Devido ao seu tamanho pequeno, é difícil estudar as jangadas lipídicas in vivo. Uma forma útil para estudar o papel de jangadas lipídicas é alterar os seus constituintes. Metil-β-ciclodextrina (MβCD) é um composto que foi encontrado para esgotar o colesterol das membranas de mamíferos e é normalmente utilizado para estudar o papel das jangadas lipídicas 18. Neste protocolo, que apresentam um método para esgotar o colesterol das membranas das células hospedeiras e quantificar o efeito do esgotamento da capacidade das células hospedeiras para fagocitar C. neoformans in vitro. Este procedimento faz uso de cultura de células techniques sobre um macrófago imortalizado como linha celular (J774A.1) como um modelo para a infecção. Depleção de colesterol foi realizada por exposição a MβCD, que tem um núcleo hidrofóbico específico para o tamanho de esteróis e é capaz de actuar como um dissipador para o colesterol desenhá-lo para fora da membrana 19. Depleção de colesterol foi medida quantitativamente usando um kit disponível comercialmente e qualitativamente utilizando uma extracção de lípidos Bligh-Dyer modificado, seguidas por cromatografia em camada fina (TLC) 20. . Fagocitose foi medida através da infecção da linha celular com uma cultura de fermento opsonizado misturado com um cocktail de interferão-γ e lipopolissacárido para activar os macrófagos Cryptococcus foi opsonizadas utilizando um anticorpo glucuronoxylomannan (GXM) 21-23. A coloração e microscopia de experiências permitiu a visualização de células e do cálculo do índice de fagocitose de avaliar o grau de fagocitose. Tomados em conjunto, este protocolo describes um método de base que integra a alteração da composição lipídica com um processo fisiológico.

<table border="0" cellpadding="0" cellspacing="0" width="622">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:120px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Class II type A2 Biosafety Cabinet</td>
			<td style="width:120px;">Labconco</td>
			<td style="width:119px;">3460009</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">J774A.1 cell line</td>
			<td style="width:120px;">ATCC</td>
			<td style="width:119px;">TIB-67</td>
			<td style="width:167px;">Arrives Frozen. See ATCC instructions for culturing.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">11995-065</td>
			<td style="width:167px;">Store at 4 &#176;C and warm to 37 &#176;C prior to use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">HI Fetal Bovine Serum Performance Plus</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">10082-147</td>
			<td style="width:167px;">Keep frozen at -20 &#176;C and thaw before adding to DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;">Used to suplement DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isotemp Cell culture incubator</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model # 3530</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well culture dish</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3595</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x Phosphate Buffered Saline</td>
			<td style="width:120px;">Fisher Scientific BioReagents</td>
			<td style="width:119px;">BP3994</td>
			<td style="width:167px;">Dilute to 1x and filter or autoclave prior to use.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Methyl-&#946;-Cyclodextrin</td>
			<td style="width:120px;">Sigma Life Science</td>
			<td style="width:119px;">C4555-10G</td>
			<td style="width:167px;">Dissolve in 1x PBS to make solutions of 10mM and 30mM concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Orbital Shaker</td>
			<td style="width:120px;">Labline</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Amplex Red Cholesterol Assay Kit</td>
			<td style="width:120px;">Life Technologies Molecular Probes</td>
			<td style="width:119px;">A12216</td>
			<td style="width:167px;">All reagents for Cholesterol Assay are contained within the kit. Follow Manufacturer instructions.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well Black Assay plate</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3603</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FilterMax microplate reader</td>
			<td style="width:120px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model F5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TLC Chamber</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z126195-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">650498-4L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">34860-2L-R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TLC Paper</td>
			<td style="width:120px;">Whatman</td>
			<td style="width:119px;">3030917</td>
			<td style="width:167px;">Cut down to size needed for TLC tank</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fume Hood</td>
			<td style="width:120px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Any fume hood that complies with AIHA/ANSI Standards&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">6-Well Plate</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3506</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Scraper</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hemocytometer&#160;</td>
			<td style="width:120px;">Hausser Scientific</td>
			<td style="width:119px;">1490</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:120px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model Alegra x-30R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Votex Mixer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:119px;">12-812</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Balance</td>
			<td style="width:120px;">Mettler Toledo</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model # MS104S Meaures down to .1 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Pasteur Pipette</td>
			<td style="width:120px;">Fisherbrand</td>
			<td style="width:119px;">13-678-20A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cholesterol</td>
			<td style="width:120px;">Avanti Polar Lipids</td>
			<td style="width:119px;">700000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SpeedVac Concentrator</td>
			<td style="width:120px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model # SPD2010&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petroleum Ether</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:119px;">E139-1&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl Ether</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">309966&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetic Acid</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">320099</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">TLC Silica Gel 60 with concentrating zone</td>
			<td style="width:120px;">Analytical Chromatograhy Millipore</td>
			<td style="width:119px;">1.11845.0001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodine Chips</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">376558-50G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sulfuric Acid</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">320501&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Manganese Chloride</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">244589</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">UVP EC3 Imaging System</td>
			<td style="width:120px;">Ultra-Violet Products Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Use the Vision Works LS software for densitometry analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Bottom Confocal Dish</td>
			<td style="width:120px;">MatTek</td>
			<td style="width:119px;">P35G-1.5-10C</td>
			<td>www.glassbottomdishes.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cryptococcus neoformans (H99)</td>
			<td style="width:120px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Obtained from Duke University Medical Center</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">YNB</td>
			<td style="width:120px;">BD</td>
			<td style="width:119px;">239210</td>
			<td style="width:167px;">See manufacturer for preparation instructions. Use a Glucose concentration of 20 g/L.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Lipopolysaccharide</td>
			<td style="width:120px;">Sigma</td>
			<td style="width:119px;">L4391-1MG</td>
			<td style="width:167px;">Dissolve in 1x PBS to make 1mg/mL stock. Store at -20 &#176;C.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Interferon gamma</td>
			<td style="width:120px;">Sigma</td>
			<td style="width:119px;">I4777</td>
			<td style="width:167px;">Dissolve in 1x PBS to make .1 mg/mL stock solution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Glucuronoxylomannan antibody (anti-GXM)</td>
			<td style="width:120px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gift from Arturo Casadevall&#39;s Lab concentration is 1.98 mg/mL</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">Giemsa</td>
			<td style="width:120px;">MP Biomedicals</td>
			<td style="width:119px;">194591</td>
			<td style="width:167px;">Dissolve .8 g of Giemsa in 25 mL of Glycerol and heat to 60 &#176;C for 1 hour. Add 25 mL of methanol to the solution and allow to age at room temperature for at least 1 month.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Microscope</td>
			<td style="width:120px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Observer.D1 microscope with AxioCam MRm for taking images</td>
		</tr>
	</tbody>
</table>

macrophage cholesterol depletion and its effect on the phagocytosis of cryptococcus neoformans

A criptococose é uma infecção potencialmente fatal causada por fungos patogênicos do gênero Cryptococcus. A infecção ocorre quando da inalação de esporos, que são capazes de replicar no pulmão profundo. A fagocitose por macrófagos de Cryptococcus é uma das formas que a doença é capaz de se espalhar para o sistema nervoso central para causar meningoencefalite letal. Portanto, o estudo da associação entre Cryptococcus e macrófagos é importante para entender a progressão da infecção. O presente estudo descreve um protocolo passo-a-passo para estudar macrófagos infecciosidade por C. neoformans in vitro. Usando este protocolo, o papel de esteróis de acolhimento em interações patógeno-hospedeiro é estudado. Diferentes concentrações de metil - ciclodextrina (MCD) foram usadas para esgotar o colesterol do sarcoma murino retículo semelhantes a macrófagos linha celular J774A.1. Depleção de colesterol foi confirmada e quantificada utilizando tanto um dis comercialmentee kit de colesterol quantificação e cromatografia em camada fina. Células de colesterol empobrecido foram activadas utilizando lipopolissacarídeo (LPS) e interferão gama (IFNy) e infectados com opsonizadas com anticorpos Cryptococcus neoformans de tipo selvagem em células H99 uma razão efector-para-alvo de 1: 1. As células infectadas foram monitorados após 2 horas de incubação com C. neoformans e seu índice fagocitário foi calculado. Depleção de colesterol resultou numa redução significativa do índice de fagocitose. Os protocolos apresentados oferecer um método conveniente para imitar o início do processo de infecção em ambiente de laboratório e estudar o papel da composição lipídica alojamento na infectividade.

Colesterol Esgotamento Análise do sobrenadante reservado no passo 1.3 do protocolo, seguindo as instruções do fabricante no kit de Ensaio de Colesterol Amplex Red produz uma concentração elevada de colesterol na amostra MβCD tratados em comparação com o controlo de PBS 1x. Dependendo do tipo de célula e a depleção de colesterol concentração MβCD utilizada pode variar. Para J774 tratados com 10 mM de MβCD, foi observada uma diminuição de aproximadamente 50%. A depleção pode se...

Watch this Scientific Journal Video about Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans at JoVE.com

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Macrófago Colesterol Exaustão e seu efeito sobre a fagocitose de<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

macrophage-cholesterol-depletion-its-effect-on-phagocytosis

Research

JoVE Journal

Immunology and Infection

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

12.6K visualizações.  Stony Brook University. In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections. 

Vídeo: Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Esgotamento das populações de células específicas de Esgotamento Complemento

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Imunologia e Infecção

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Depleção e reconstituição de macrófagos em Ratos

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

O efeito sinérgico da luz visível e no Gentamicina<em&gt; Pseudomonas aeruginosa</em&gt; Microorganismos

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Usando Click Chemistry medir o efeito da infecção viral sobre a síntese de RNA do Host-Cell

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

A visualização não-lítica exocitose de<em&gt; Cryptococcus neoformans</em&gt; De macrófagos Usando Digital Microscopia de Luz

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Técnica High Throughput Fluorometric de Avaliação da macrófago fagocitose e actina Polimerização

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Transformação Biolistic de uma fluorescente Tagged Gene para o oportunista fúngica Pathogen<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

A purificação a partir de ARN intracelularmente Grown<em&gt; Listeria monocytogenes</em&gt; Em macrófagos

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Avaliação dos efeitos de drogas baseados em anticorpo murino da medula óssea e ativação de macrófagos peritoneais

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

Size Matters: Medição do diâmetro da cápsula em Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

Macrófago Colesterol Exaustão e seu efeito sobre a fagocitose de

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