Este trabalho foi apoiado pelo National Institutes of Health / Instituto Nacional de Distúrbios Neurológicos e Derrame (NIH / NINDS) concede R01-NS074387, R01-R21 NS057711 e-NS091555 para MGC; NIH / NINDS concede R01-NS061107, R01-NS076991, R01 e R21-NS082311-NS084275 a PRL; subvenções de corações felizes de Leah, da Universidade de Michigan Comprehensive Cancer Center atribuído a MGC e PRL; o Departamento de Neurocirurgia da Universidade de Michigan School of Medicine; do Instituto Michigan de Pesquisa Clínica e da Saúde, financiado pelo NIH concessão 2UL1-TR000433; a bolsa de formação Biologia da Universidade de Michigan Cancer financiado pelo NIH / NCI (National Cancer Institute) concessão T32-CA009676; da Universidade de Michigan Treinamento em clínica e básica Neuroscience suportado pelo NIH / NINDS conceder T32-NS007222; e da Universidade de Programa de Formação Médica Cientista Michigan financiado pelo NIH / NIGMS (Instituto Nacional de Medicina Ciências Gerais) conceder T32-GM007863. Os autores de umre grato pela liderança acadêmica e apoio recebido do Dr. Karin Muraszko e do Departamento de Neurocirurgia; a M. Dahlgren, D. tomford, e S. Napolitan para apoio administrativo soberba; a M. Dzaman para a assistência técnica excelente; e Phil F. Jenkins para apoio generoso para a compra de um Microscópio Eletrônico de Varredura Zeiss 3D. Reconhecemos também o laboratório Kuchroo na Harvard Medical School a partir do qual uma versão modificada da estratégia mediada por meios centrifugação de densidade para isolamento de células mononucleares do cérebro foi desenhado.

<ol>
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Este protocolo descreve um método robusto e reprodutível para o isolamento e análise de citometria de fluxo de PBMC que se infiltraram no rato precoce microambiente do tumor cerebral. As suspensões de células de glioma são gerados a uma concentração especificada pelo experimentalista que são enxertados estereotaxicamente no corpo estriado do cérebro do rato. Os ratos são então sacrificados a pontos de tempo pré-determinados especificados pelo desenho experimental e os seus cérebros são colhidas e transfor...

Os gliomas são uma classe de cancros cerebrais gliais que derivam de células gliais transformadas dentro do sistema nervoso central (SNC). De todos os gliomas, Organização Mundial da Saúde (OMS) glioma grau IV ou glioblastoma (GBM), é o mais comum e letal 1. GBM é altamente refractário ao tratamento padrão-de-curso que consiste na ressecção do tumor na medida do possível, seguida por radiação, mais quimioterapia concomitante e adjuvante com temozolomida 2. Estes cancros mortais transportar um prognóstico sombrio de apenas 15-18 meses de sobrevivência a partir do momento do diagnóstico inicial com apenas 5% dos pacientes que sobrevivem a doença depois de 5 anos 3. A presença da barreira hematoencefálica (BHE), a falta de células apresentadoras de antigénios profissionais (APCs), e a existência de estruturas previamente não identificado linfáticos bona fide dentro do cérebro 4 conduziram à noção de GBM como imune privilegiada. No entanto, numerosos estudos agora shoW que estes cancros cerebrais, de facto geram o recrutamento de células imunitárias periféricas que são predominantemente de origem mielóide, que incluem monócitos, macrófagos e células supressoras derivadas de mielóides (MDSCs) 5. GBM também influencia a atividade de microglia cérebro-residente para se tornar pró-tumorigenic 6,7. As células linfóides, tais como as células T CD8 + 8 e células CD56 + assassinas naturais 9 também estão presentes dentro do microambiente do tumor, mas em muito menor número, um facto provavelmente devido à função imunossupressora instigada por factores derivadas de glioma em macrófagos associados a tumores ( TAM) 10. Células T CD4 + também estão presentes no GBM, mas grande parte dessa população também expressa CD25 e FoxP3, fabricantes de imunossupressora T reguladoras (reg T) as células 11. O estado imunossupressora total do GBM culmina com a promoção da fuga imunológica e progressão tumoral 12. <p class= &quot;jove_content&quot;&gt; Uma melhor compreensão dos mecanismos de GBM imunossupressão é crítico para o desenvolvimento de estratégias eficazes imunoterapêuticas concebidas para estimular o sistema imunitário contra o tumor. Ao longo dos últimos 15 anos, nosso laboratório tem trabalhado para superar os mecanismos do cérebro immunosuppresson tumor, a fim de desenvolver novas immunotherapeutics eficazes anti-GBM 13-19. O culminar deste trabalho tem levado a um ensaio clínico destinado a avaliar um citotóxico combinada e terapêutica imuno-estimulante para os pacientes com diagnóstico recente de GBM (ClinicalTrials.gov Identificador: NCT01811992). O nosso trabalho mais recente mostra que o GL26 de ratinho e rato de CNS-1 células GBM bloquear antitumoral vigilância imunitária das células NK através da produção de grandes quantidades de a lectina de ligação a β-galactósido galectina-1 (Gal-1) 20. Isto foi demonstrado através da supressão da expressão de Gal-1 em células de glioma de knockdown utilizando shRNA gene mediada. In vitro experiments mostraram que as células de glioma Gal-1 deficiente proliferaram normalmente na cultura, no entanto, sofreu rejeição rápida logo após o enxerto intracraniana em singï¿½ico C57BL / 6J ou RAG1 - / - ratos, estabelecendo assim a independência do T ou células B sobre esta forma de rejeição de tumor. Imunodepleção de células NK com anticorpos anti-asialo GM-1 anti soro ou anticorpos monoclonais NK1.1 conduziram à recuperação completa do crescimento de glioma Gal-1-deficiente intracraniana, estabelecendo o papel das células NK em Gal-1-deficiente rejeição glioma. Mostramos agora que imunodepleção de células mielóides Gr-1 + / CD11b + é suficiente para impedir que o glioma rejeição Gal-1-deficiente apesar da presença de células NK, revelando assim um papel auxiliar indispensável para células mielóides no favorecimento de Gal NK mediada lise tumoral -1 com deficiência (dados não publicados). Este resultado inesperado nos levou a desenvolver um protocolo abrangente para o isolamento e análise de células mononucleares do sangue periférico (PBMC), queinfiltrar no microambiente do tumor cerebral logo após o enxerto intracraniana para que possamos melhor caracterizar os eventos de infiltração do sistema imunológico que predicado glioma rejeição gal-1-deficiente. O método é demonstrado aqui, usando células de glioma do rato GL26 que a proteína fluorescente mCitrine constitutivamente expresso, chamado GL26-Cit, que permitem a visualização de células tumorais direta por microscopia de fluorescência 21. Estas células são enxertados estereotacticamente no cérebro de singeneicos C57BL / 6J e são deixadas a crescer durante 24, 48, ou 72 horas antes do rato eutanásia. PBMC infiltrantes de glioma são então isolados e immunolabeled usando anti -CD45, -GR-1, -CD11b -NK1.1 e anticorpos de superfície da célula, juntamente com imunomarcação intracelular para a granzima B (GzmB). Esta combinação específica de anticorpos permite a identificação de Gr-1 + / + CD11b células mielóides infiltrantes tumorais e NK1.1 +, células NK, os tipos de células que temos been implicados na rejeição de tumor Gal-1-deficiente. O perfil imunológico infiltração de Gal-1-deficiente GL26-Cit glioma, referido aqui como GL26-Cit-gal1i, é então comparada com a de gliomas que expressam níveis normais de Gal-1 chamado GL26-Cit-NT que contêm uma não- segmentação controle hairpin shRNA. O protocolo começa com uma descrição de como a cultura de células de glioma GL26-Cit in vitro, que é seguido por uma explicação sobre como ortotopicamente enxertar essas células para o corpo estriado de singï¿½icos camundongos C57BL / 6J. Em seguida, prossegue para enumerar as etapas envolvidas no isolamento e imunomarcação de PBMCs infiltrantes de glioma para análise de citometria de fluxo. O protocolo termina com uma explicação de análise de dados padrão e representação gráfica. A demonstração revela que tanto a Gr-1 + / + CD11b células mielóides e células NK NK1.1 + preferencialmente se acumular dentro do microambiente do tumor cerebral Gal-1-deficiente dentro 48hr de implantação do tumor, um resultado que ajuda a explicar por que esses tumores rapidamente sofrer lise tumoral completa aproximadamente 1 semana enxerto pós-tumoral 20. O método é facilmente adaptável a um número de diferentes modelos experimentais in vivo em que os dados temporais de infiltração imune no cérebro é necessária. Uma única experimentalista podem executar o protocolo de colheita cérebro para análise citométrica de fluxo de PBMCs infiltrantes de glioma em cerca de 4-6 horas, dependendo do número de amostras a serem analisadas. O método pode também ser combinado com experiências destinadas para caracterizar o perfil de PBMCs em circulação em ratinhos portadores de tumor em comparação com aqueles que se infiltrar no cérebro, de forma a identificar os fenótipos imunossupressão especificamente induzidas pelo microambiente do tumor. Aplicação desta e de semelhantes métodos deverá facilitar uma melhor compreensão dos fatores envolvidos no tráfico de células imunes periféricas no microambiente do tumor cerebral.

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	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modification of Eagles Medium</td>
			<td>Gibco</td>
			<td>12430-054</td>
			<td>with 4.5g/L D-glucose, L-glutamine, 25mM HEPES and phenol red&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-buffered Saline&#160;</td>
			<td>Gibco</td>
			<td>14190-144&#160;</td>
			<td>without calcium chloride or Magnesium chloride</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Omega Scientific Inc.</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>10,000U/ml Penicillin; 10,000&#956;g/ml Streptomycin&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>200mM</td>
		</tr>
		<tr>
			<td>G418 sulfate&#160;</td>
			<td>Omega Scientific Inc.</td>
			<td>GN-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>from Streptomyces alboniger</td>
		</tr>
		<tr>
			<td>HyClone HyQTase non-mammalian trypsin alternative&#160;</td>
			<td>Thermo Scientific</td>
			<td>SV30030.01</td>
			<td>in DPBS with EDTA&#160;</td>
		</tr>
		<tr>
			<td>Phase contrast hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>0.1mm deep</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 0.9% NaCl injection, USP</td>
			<td>Hospira</td>
			<td>NDC: 0409-4888</td>
			<td>10ml vials</td>
		</tr>
		<tr>
			<td>0.6ml conical polypropylenemi microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezole hydrochloride injection</td>
			<td>Orion Pharma</td>
			<td>NDC: 52483-6298</td>
			<td>5mg/ml solution</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection</td>
			<td>Fort Dodge</td>
			<td>NDC: 0409-2051</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Dexmedetomidine hydrochloride injection</td>
			<td>Zoetis</td>
			<td>NDC: 54771-2805</td>
			<td>0.5mg/ml solution</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride injection</td>
			<td>Lloyd</td>
			<td>NDC: 61311-481</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Carprofen injection</td>
			<td>Pfizer</td>
			<td>NDC: 61106-8501</td>
			<td>50mg/ml solution</td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride injection</td>
			<td>Reckitt Benckiser</td>
			<td>NDC: 12496-0757</td>
			<td>0.3mg/ml ampuls</td>
		</tr>
		<tr>
			<td>1ml tuberculin syringes&#160;</td>
			<td>Covidien</td>
			<td>8881501400</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x &#189; (0.45mm x 13mm) syringe needles&#160;</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical clippers</td>
			<td>3M</td>
			<td>9661</td>
			<td></td>
		</tr>
		<tr>
			<td>Providone-iodine solution</td>
			<td>Aplicare</td>
			<td>82-217</td>
			<td>NDC: 52380-1905</td>
		</tr>
		<tr>
			<td>Sterile petrolatum ophthalmic ointment&#160;</td>
			<td>Dechra</td>
			<td>NDC: 17033-211</td>
			<td></td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol prep pads</td>
			<td>Kendall</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Covidien</td>
			<td>8044</td>
			<td>non-woven, 4&#34; x 4&#34;, 4-ply&#160;</td>
		</tr>
		<tr>
			<td>1.7ml conical polypropylene microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-281</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse stereotactic frame</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical lamp</td>
			<td>Philips Burton</td>
			<td>CS316W</td>
			<td>Coolspot II Variable Spotlight</td>
		</tr>
		<tr>
			<td>Curved dissecting forceps</td>
			<td>Ted Pella Inc.</td>
			<td>5431</td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri retractors&#160;</td>
			<td>FST</td>
			<td>17000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless precision power drill</td>
			<td>Dremel&#160;</td>
			<td>1100-01</td>
			<td>Stylus model; 7.2-Volt Lithium-Ion with Docking Station</td>
		</tr>
		<tr>
			<td>Engraving Cutter</td>
			<td>Dremel</td>
			<td>105</td>
			<td>1/32&#34; or 0.8 mm bit diameter</td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>75</td>
			<td>Hamilton Microliter&#174; Syringes 700 series; 5&#956;l volume</td>
		</tr>
		<tr>
			<td>Microliter syringe needles</td>
			<td>Hamilton</td>
			<td>7762-06</td>
			<td>33G, small hub RN NDL, 1.5 in, point style 3, 6/PK</td>
		</tr>
		<tr>
			<td>Ethilon 3-0 black monofilament nylon sutures&#160;</td>
			<td>Ethicon</td>
			<td>1663</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bar</td>
			<td>VWR</td>
			<td>74950-296</td>
			<td>7.9mm diameter x 50mm length (5/16&#34; diameter x 2&#34; length)</td>
		</tr>
		<tr>
			<td>1L glass screw-cap storage bottle</td>
			<td>Corning</td>
			<td>1395</td>
			<td>Type 1, Class A borosilicate glass</td>
		</tr>
		<tr>
			<td>Heparin sodium&#160;</td>
			<td>Sagent</td>
			<td>NDC: 25021-400</td>
			<td>1,000U/ml solution</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Cole Parmer Instruments Group</td>
			<td>77200-60</td>
			<td>Master Flex Easy Load II console drive</td>
		</tr>
		<tr>
			<td>compressed carbogen (95% O2 / 5% CO2)</td>
			<td>available from local vendor</td>
			<td>N/A</td>
			<td>A-type, large cylinder&#160;</td>
		</tr>
		<tr>
			<td>20G x 1 &#189;&#34; aluminum hub blunt needles</td>
			<td>Kendall</td>
			<td>8881202363</td>
			<td>0.9mm x 38.1mm</td>
		</tr>
		<tr>
			<td>Polyurethane ice bucket</td>
			<td>Fisherbrand</td>
			<td>02-591-45</td>
			<td>Capacity: 0.152 oz. (4.5L)</td>
		</tr>
		<tr>
			<td>Collagenase (Type I-S)</td>
			<td>Sigma Aldrich</td>
			<td>C1639</td>
			<td>from Clostridium histolyticum&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS002007</td>
			<td>&#62;2,000 Kunitz units per mg dry weight</td>
		</tr>
		<tr>
			<td>Antistatic polystyrene hexagonal weighing dishes</td>
			<td>Ted Pella Inc.</td>
			<td>20157-3</td>
			<td>top I.D. 115mm, base I.D. 85mm, 203ml volume&#160;</td>
		</tr>
		<tr>
			<td>Stainless steel single edged razor blades&#160;</td>
			<td>Garvey</td>
			<td>40475</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone rongeurs</td>
			<td>FST</td>
			<td>16001-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>FST</td>
			<td>13014-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Large dissection scissors</td>
			<td>Ted Pella Inc.</td>
			<td>1316</td>
			<td></td>
		</tr>
		<tr>
			<td>Small dissection scissors</td>
			<td>FST</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end forceps</td>
			<td>Ted Pella Inc.</td>
			<td>13250</td>
			<td></td>
		</tr>
		<tr>
			<td>7ml glass Dounce tissue grinder</td>
			<td>Kontes</td>
			<td>KT885300-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll Density Centrifugation Media&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>Genesee Scientific</td>
			<td>12-104</td>
			<td>single use; sterile</td>
		</tr>
		<tr>
			<td>70&#956;m sterile nylon mesh cell strainers&#160;</td>
			<td>Fisherbrand</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11)</td>
			<td>Biolegend</td>
			<td>103128</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti-mouse NK1.1 (clone: PK136)</td>
			<td>eBiosciences&#160;</td>
			<td>17-5941-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5)</td>
			<td>BD Pharmigen</td>
			<td>553128</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70)&#160;</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400628</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG2a, &#954; (clone: eBM2a) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>17-4724</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat IgG2b, &#954; (clone: eB149/10H5) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>12-4031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control</td>
			<td>Biolegend</td>
			<td>400632</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse anti-mouse granzyme B (Clone: GB11)&#160;</td>
			<td>Biolegend</td>
			<td>515403</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse IgG1, &#954; (Clone: MOPC-21) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400151</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm</td>
			<td>BD</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-103</td>
			<td>conical bottom&#160;</td>
		</tr>
		<tr>
			<td>50ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td>conical bottom</td>
		</tr>
		<tr>
			<td>12x75mm round-bottom polypropylene FACS tubes&#160;</td>
			<td>Fisherbrand</td>
			<td>14-956-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria Special Order Research Product flow cytometer/cell sorter</td>
			<td>BD&#160;</td>
			<td>650033</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II Biological Safety Cabinet</td>
			<td>The Baker Company</td>
			<td>SG603</td>
			<td>model: SterilGARD III Advance</td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells

Nosso laboratório tem demonstrado recentemente que natural killer (NK) são capazes de erradicar GL26 rato ortotopicamente implantado e rato CNS-1 gliomas malignos logo após o enxerto intracraniana se as células cancerosas se tornam deficientes em sua expressão da galectina lectina β-galactoside de ligação -1 (Gal-1). Um trabalho mais recente mostra agora que uma população de células mielóides + / CD11b + GR-1 é essencial para este efeito. Para melhor compreender os mecanismos pelos quais NK e células mielóides cooperam para conferir rejeição tumoral Gal-1 deficiente em temos desenvolvido um protocolo detalhado para o isolamento e análise de células mononucleares de sangue periférico que se infiltram no glioma (PBMC). O método é demonstrado aqui pela comparação PBMC infiltração no microambiente tumoral de Gal-1 que expressam GL26 gliomas com aqueles preparados Gal-1-deficiente através shRNA knockdown. O protocolo começa com uma descrição de como a cultura e preparar GL26 cells para inoculação no singï¿½ico C57BL / 6J cérebro do rato. Em seguida, explica as etapas envolvidas no isolamento e análise de citometria de fluxo de PBMCs infiltrantes glioma do microambiente do tumor cerebral precoce. O método é adaptável a um número de modelos experimentais in vivo em que os dados temporais de infiltração imune no cérebro é necessária. O método é sensível e altamente reprodutível, como PBMCs infiltrantes glioma pode ser isolado a partir de tumores intracranianos, logo que 24 hr pós-enxerto de tumor com contagens de células semelhantes observadas em tumores de pontos de tempo ao longo combinado experiências independentes. Uma única experimentalista pode executar o método de colheita cérebro para análise citométrica de fluxo de PBMCs infiltrantes de glioma em cerca de 4-6 horas, dependendo do número de amostras a serem analisadas. Modelos de glioma alternativos e / ou de detecção de anticorpos específicos de células também podem ser utilizados à discrição dos experimentalistas para avaliar a infiltração de vários outros Immune tipos de células de interesse, sem a necessidade de alterações no procedimento geral.

A estratégia de gating seguinte é usada para uma experiência típica: FSC vs. SSC-A-A → SSC-H vs. SSC-W → FSC-H vs. FSC-W → CD45 vs. contagem → Gr-1 vs. CD11b → NK1.1 vs. contagem. Gates, colocado em Gr-1 + / + CD11b células mielóides e células NK NK1.1 +, em seguida, são estratificados com base na expressão GzmB (Figura 5A). Backgating células identificadas como células NK NK1.1 + e Gr-1 + / + CD11b células mielóide...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Apresentado aqui é um método simples para o isolamento e análise de citometria de fluxo de células mononucleares de sangue periférico que se infiltram no glioma que produz dados quantitativos tempo-dependente do número e estado de activação das células do sistema imunológico que entram no microambiente do tumor cerebral precoce.

Isolamento e Análise de Citometria de Fluxo infiltrantes Glioma Células Mononucleares do Sangue Periférico

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

isolation-flow-cytometric-analysis-glioma-infiltrating-peripheral

Research

JoVE Journal

Immunology and Infection

15.6K visualizações.  University of Michigan.  Apresentado aqui é um método simples para o isolamento e análise de citometria de fluxo de células mononucleares de sangue periférico que se infiltram no glioma que produz dados quantitativos tempo-dependente do número e estado de activação das células do sistema imunológico que entram no microambiente do tumor cerebral precoce. 

Vídeo: Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Isolamento de células de camundongo Peritoneal Cavity

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Imunologia e Infecção

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Detecção de glicogênio no sangue periférico células mononucleares com Periodic Acid Schiff Coloração

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e análise de citometria de fluxo de células imunes do cérebro isquêmico Rato

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Citometria de fluxo e assassinas naturais celular Lytic Actividade em sangue total humano

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolamento e Citometria de Fluxo Analysis of Human Endocervical Gama Cells Delta T

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Digestão do fígado murino para um fluxo Cytometric análise de células endoteliais linfáticas

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

Digestão de olhos inteiros de rato para análise citométrica de fluxo multi-parameter de fagocitos mononucleares

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Isolamento, ativação e expansão de células T baseadas em flotação de amostras de células mononucleares do sangue periférico humano usando microbolhas

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Coleta e crioarmazenamento de PBMC para análise bioenergética mitocondrial

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Autor em destaque: Preservação de parâmetros bioenergéticos em células mononucleares do sangue periférico após criopreservação 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

Criopreservação e Avaliação Bioenergética de Células Mononucleares do Sangue Periférico Humano

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...