Kanada Sağlık Araştırma, Lösemi ve Lenfoma Derneği Kanada ve Kanada Lenfoma Vakfı Enstitüleri hibe ile desteklenir.

Reaktifler hazırlayın Not: Tüm PBS içeren Ca 2 + ve Mg 2 + <ol><li> Algılama Reaktif Oda sıcaklığında PBS 20μM 7-amino-4-klorometil kumarin, t-BOC-Leucine-metiyonin amid (BOC-LM-CMAC) ekleyin. Her bir hücre süspansiyonu 2mL algılama reaktif gerekecektir. </li><li> % 1 Paraformaldehit (Sabit Hücreler) Oda sıcaklığında PBS içinde% 4 paraformaldehid stok çözeltinin. % 1 ml 1 paraformaldehid her FACS tüp kısım. Üç FACS tüpleri her deneysel koşul için gereklidir (örneğin 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 9 FACS tüpleri gerekir) </li><li> PD150606 (calpain inhibitörü) 100 mM nihai konsantrasyonu DMSO içinde sulandırın PD150606. Bu reaktif ve bu deney süresi boyunca ışık ile tedavi edilen tüm hücreleri koruyun. </li><li> PD98059 (MEK1 inhibitörü) 50mm son konsantrasyonu DMSO sulandırın PD98059. </li></ol> Hücreler hazırlayın <ol><li> 5 x 10 Opti-MEM medya ml başına 1 ila 5 x 10 6 hücre% 5 fetal sığır serumu, WEHI-3, IL-3 kaynağı (IL-3 gibi herhangi bir başka kaynak olarak süpernatant ile desteklenmiş 32Dkit hücrelerin büyümesine rekombinant protein gibi),% 0.1 &#39;i 2-merkaptoetanol ve antibiyotikler. </li><li> Üç 15 mL Falcon tüpleri (tüp başına 4 x 10 6 hücre), 12 x 10 6 32Dkit hücreleri toplayın . </li><li> 15 yaşında 5 dakika boyunca 1000 RPM Pelet hücreleri ° C </li><li> Süpernatant aspire. 2mL oda sıcaklığında PBS içinde hücreler tekrar </li><li> 50μM PD150606 bir hücre süspansiyonu davranın. Örnek Falcon tüp alüminyum folyo ile kaplayarak ışıktan korumalı olduğundan emin olun. Vortex karanlıkta, oda sıcaklığında kısa bir süre sonra yirmi ila otuz dakika boyunca inkübe edin. </li><li> 20μM PD98059 ile ikinci hücre süspansiyonu davranın. Plaka 37, 2 saat boyunca 35mm doku kültürü çanak ve inkübe hücrelerin ° C </li></ol> Sabit Hücreler Calpain Testi <ol><li> Örnekleri PD150606 kuluçka calpain tahlil, tedavi edilmeyen numuneler üzerinde başlar. Her hücre süspansiyonu 2mL algılama reaktif ekleyerek başlayın. % 1 paraformaldehid (0 dakika zaman noktası) ile hemen önceden hazırlanmış FACS tüpler için 1 ml hücre süspansiyonu / algılama reaktif karışımı ekleyin. </li><li> Hücre süspansiyonları oda sıcaklığında 5 dakika boyunca algılama reaktifi ile inkübe edin. Daha sonra 1 ml% 1 paraformaldehid ile hücre süspansiyonu FACS tüpüne 1 ml ekleyin. </li><li> Algılama karışımı ile hücre süspansiyonları ek bir 5 dakika oda sıcaklığında inkübe devam edin. Daha sonra 1 ml% 1 paraformaldehid ile hücre süspansiyonu FACS tüpüne 1 ml ekleyin. </li><li> Inhibitörleri ile tedavi edilmiştir 32Dkit hücreleri calpain tahlil tekrarlayın. </li><li> Fix karanlık gecede hücreleri 4 ° C </li><li> Ertesi gün pelet 5 dakika boyunca 1800 RPM hücreleri 15 ° C </li><li> Süpernatant aspire. 750uL PBS hücrelerin yeniden süspanse edin. Buz üzerinde hücrelerini koyun ve karanlıkta tutmak. </li></ol> Sabit Hücreleri için Veri Edinme <ol><li> LSR II (BD Bioscience) kullanarak hücrelerin analiz edin. Filtreler Lightwave Xcyte (UV) lazer, uyarma hattı 355nm ve lazer gücü 25mW, substrat ve ürün için sırasıyla 405 ve 450 nm emisyon için ayarlanır. Substrat ve ürün Band geçiren filtreler, 405/20 ve 450/50. Uzun geçiren filtreler substrat ve ürün için 405 ve 450. PMT gerilim ürün için substrat için 350-375 ve 325-350 kurulmuştur. </li><li> Yan dağılım; Hint-1 Mavi (log); Mor Hint-1 (log) forward scatter: Aşağıdaki parametreler ile BD FACSDiva deney set-up. Forward scatter canlı hücreler üzerinde kapısı (Şekil 1a), Hint-1 Mavi histogram, Hint-1 Mor histogram, Hint-1 Mavi vs Hint-1 Menekşe ile yan dağılım nokta arsa vs: Çalışma sayfasında aşağıdaki grafikler nokta arsa. </li><li> LSR AlignFlow ve AlignFlow II artı floresan boncuk (Invitrogen) kalibre edin. PMT gerilim her deneyde önce aynı kanalda boncuk floresan histogramın zirvesine yerleştirmek için ayarlanabilir olmalıdır. </li><li> 0 dakika zaman noktada 32Dkit hücreleri kullanarak veri edinme başlayın. Gerekli parametre gerilimler ayarlayın. Edinin ve örnek başına 10.000 olayları kaydetmek. </li><li> Her tüp arasındaki FACS tamponu ile makine durulama, her bir numune için elde tekrarlayın. </li><li> İhracat verileri. Emin olun LSR II enstitü kurallarına göre temizlenir. </li></ol> Canlı Hücrelerde Calpain Testi <ol><li> Hiçbir PD150606, yukarıdaki gibi hücre süspansiyonları hazırlayın. LSR II örnekleri getirin ve yukarıdaki gibi filtreler ve parametrelerini ayarlamak. Yukarıdaki gibi çalışma sayfası ayarlayın. Hint-1 Mavi vs gösteren büyük bir nokta arsa dahil. Maksimum sayı elde etmek için programı ayarlayabilirsiniz.olaylar ber (durak kapısı). </li><li> 32Dkit hücreleri 50μM PD150606 bir hücre süspansiyonu ekleyin. Karanlıkta örnek tutun ve 20-30 dakika oda sıcaklığında inkübe edin. </li><li> Hiçbir PD150606 32Dkit hücre süspansiyonu elde başlayın. Düşük akış hızı saniyede 200-300 olayların kullanın. Tüp 30 saniye ile 1 dakika sonra makineden çıkarın. 20μM BOC-LM-CMAC hücrelere ekleyin. Vortex kısa bir süre sonra veri edinmeye devam. </li><li> 10-20 dakika veya calpain etkinliği yaylalarda kadar veri elde. </li><li> PD150606 ile tedavi 32Dkit hücreleri ile 3. ve 4. adımları tekrarlayın. </li><li> İhracat verileri. Emin olun LSR II enstitü kurallarına göre temizlenir. </li></ol> Kompleks (Karışık) Cep Popülasyonlar Calpain Testi * Bu deney, bir fare dalak hasat bir hücre süspansiyonu 32Dkit hücreleri belirleyecektir. <ol><li> Hasat her fare dalak. NH 4 Cl ile oda sıcaklığında 10 dakika süreyle kırmızı kan hücresi Lyse. </li><li> 2mL PBS tek hücre süspansiyonları hazırlayın. </li><li> Her iki hücre süspansiyonu bölün. 20-30 dakika karanlıkta, oda sıcaklığında 1 hücre süspansiyonu 50μM PD150606 davranın. </li><li> Sabit Hücrelerde Calpain Assay (Adımlar 1-6) için yukarıda açıklandığı gibi devam edin </li><li> Süpernatant aspire. 500μL PBS hücrelerin yıkayın. Süpernatant aspire. </li><li> % 2 BSA ile 200μL boyama tamponu hücrelerin tekrar Oda sıcaklığında 15 dakika inkübe edin. </li><li> Birincil antikor (FITC anti-fare CD117) hazırlayın. Antikor tampon boyama 1:200 dilüsyon kullanılmalıdır. Antikor karanlıkta tutulur emin olun. </li><li> Hücrelere 200μL primer antikor ekleyin. Karanlık oda sıcaklığında 30 dakika inkübe edin. </li><li> Flow sitometri için hacmini artırmak 400μL PBS ekleyin. Hücreleri buz koyun ve karanlıkta tutmak. </li><li> Sabit Hücreleri için Veri Edinme açıklandığı gibi devam edin. Parametrelere FITC ekleyin ve FITC için çalışma sayfasındaki bir histogram dahil. </li></ol> Analiz ve Temsilcisi Sonuçlar <ol><li> FlowJo (Treestar) kullanarak verileri analiz edin. Ileri ve yan dağılım profili (Şekil 1a) göre canlı hücreler üzerinde Kapısı. Canlı hücre kapısı (Şekil 1b) Hint-Blue ortalama floresan belirleyin. </li><li> Karışık Hücre Nüfus a) <ul><li> 32Dkit nüfusu tanımlamak için histogramlar ve nokta araziler FITC floresanı </li><li> Hint-Mavi 32Dkit nüfusun ortalama floresan belirleyin </li></ul></li><li> Microsoft Excel grafik ortalama floresan (Şekil 2a) kullanın. 5 ve 10 dakika arasında çizginin eğimi hesaplayın. Grafik eğimi (Şekil 2b) hücrelerinde calpain etkinliğini belirlemek için bir çubuk grafik olarak. </li></ol><img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="Şekil 1a" /> Şekil 1a ileri ve yan dağılım profiline göre canlı hücreler belirlenmesi. <img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="Şekil 1b" /> Şekil 1b 10 dakikalık bir süre boyunca ortalama floresan değişimi. <img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="Şekil 2a" /> Şekil 2a ortalama floresan Temsilcisi grafik. <img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="Şekil 2b" /> Inhibitörleri ile 32Dkit hücreler Şekil 2b. Calpain aktivite.

<ol>
	<li>Goll, D. E., Thompson, V. F., Li, H., Wei, W., Cong, 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> The Calpain System. Physiol. Rev. 83, 731-801 (2003).</li><li>Niapour, M., Berger, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometric+Measurement+of+Calpain+Activity+in+Living+Cells.">Flow Cytometric Measurement of Calpain Activity in Living Cells.</a> Cytometry A. 71A, 475-485 (2007).</li><li>Leloup, L., Daury, L., Maz&#233;res, G., Cottin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+ERK/MAP+kinase+signalling+pathway+in+milli-calpain+activation+and+myogenic+cell+migration.">Involvement of the ERK/MAP kinase signalling pathway in milli-calpain activation and myogenic cell migration.</a> The International Journal of Biochemistry and Cell Biology. 39, 1177-1189 (2007).</li></ol>

Bu video protokolü sabit ve yaşam 32Dkit hücrelerinde calpain aktivite tespit edilmiştir. Calpain inhibitörü PD150606 ile hücre hattı Tedavi, hücrelerin calpain aktivitesinin inhibisyonu ile sonuçlandı. Buna ek olarak, MEK1 inhibitörü PD98059 tedavisi, hücrelerin calpain aktivitesinin inhibisyonu ile sonuçlandı. ERK calpain-2 (3) büyük bir aktivatör. Bu sonuçlar calpain-2 aktivitesinin 32Dkit hücreleri baskın olduğunu düşündürmektedir. Laboratuvarımızda Şu çalışmalar artmış ...

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<td>PBS BOC-LM-CMAC 4% paraformaldehyde</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td> Dissolve 4g paraformaldehyde in 100mL PBS. In fume hood, heat sample with stirring until clear. Let cool then store at 4&deg;C</td>
</tr>
<tr>
<td>PD150606</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Use freshly reconstituted PD150606 for maximal calpain inhibition.</td>
</tr>
<tr>
<td>PD98059</td>
<td>&nbsp;</td>
<td>Cell Signaling Technology</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>AlignFlow, AlignFlow plus fluorescent beads FACS tubes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
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<td>LSR II</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>&nbsp;</td>
<td> Supernatant of WEHI-3 cells is used as a source of IL-3 in these experiments. Each collection of WEHI-3 supernatant is titrated to determine the optimal concentration used for 32Dkit cell growth. </td>
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measuring calpain activity in fixed and living cells by flow cytometry

Calpains her yerde hücre içi kalsiyum duyarlı, nötr sistein proteazlar<sup&gt; 1</sup&gt;. Calpains sinyalizasyon, sitoskeletal yeniden şekillenme, gen ekspresyonu, apoptozis düzenlenmesi ve hücre döngüsünün ilerlemesi de dahil olmak üzere birçok fizyolojik süreçleri, önemli rol oynarlar<sup&gt; 1</sup&gt;. Calpains kas distrofiler, kanser, diyabet, Alzheimer hastalığı ve multipl skleroz gibi birçok patolojiler suçlanmıştır<sup&gt; 1</sup&gt;. Calpain yönetmelik karmaşık ve tam olarak anlaşılamamıştır. mRNA ve protein düzeyleri calpain aktivitesini ölçmek için gen veya protein anlatım tekniklerinin kullanımı sınırlayıcı, aktivite ile zayıf ilişkilidir. Bu video protokol detayları calpain aktivitesini ölçmek için sabit ve canlı hücrelerin laboratuvar, bir akım sitometrik tahlil gelişti. Bu yöntem, calpain özellikle bölünmüş floresan substrat BOC-LM-CMAC, calpain aktivitesini ölçmek için kullanır.<sup&gt; 2</sup&gt; Bu video, sabit ve yaşayan bir fare 32Dkit lösemi hücrelerinin, tek başına veya bir splenocyte nüfusun bir parçası olarak faaliyet calpain bir LSRII (BD Bioscience) ile ölçülür. 32Dkit hücrelerin artmış aktivite normal dalak olduğu gösterilmiştir.

Watch this Scientific Journal Video about Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry at JoVE.com

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Bu makale, flow sitometri kullanarak sabit ve canlı hücrelerin calpain aktivitesini ölçmek için ayrıntılı protokol.

Sabit ve Yaşam Hücreler Sitometrisi ile Calpain Aktivitesi Ölçme

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

measuring-calpain-activity-in-fixed-and-living-cells-by-flow-cytometry

Research

JoVE Journal

Immunology and Infection

11.7K Görüntüleme.  University of Toronto. Bu makale, flow sitometri kullanarak sabit ve canlı hücrelerin calpain aktivitesini ölçmek için ayrıntılı protokol.

Video: Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Hücre-kaderi ardından<em&gt; E. coli</em&gt; Faj Lambda tarafından Enfeksiyon Sonrası

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

İmmünoloji ve Enfeksiyon

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Akış Sitometrisi ile A549 Hücreleri Eklenti ve içselleştirilmesi Gribi bir virüs Ölçme

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fare kemik iliğinden plazmasitoid dendritik hücreleri Saflaştırılması floresans ile aktive edilen hücre sınıflandırma

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Yalıtım ve fare ve insan alveoler makrofajlar Vitro kültürü

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Nötrofil kütle sitometri analizi için tüm kemik Iliği hazırlanması-Lineage hücreler

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Akış Sitometrisi Kullanılarak İnsan Lökositler tarafından Aspergillus fumigatus Conidia Fagositozunun ölçülmesi

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

Akış Sitometrisinin Kullanılmasında Eritrosit Kompleman Reseptörü 1 Ölçümü

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Akış SitometriSi-Bazlı Bir Test ile Plasmodium falciparum Parazitlerine Doğal Olarak Edinilmiş Fagositoz-Indükleyen Antikorların Ölçülmesi

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

İnsan Miyeloid U937 Hücrelerinde Akış Sitometrisi ile SAMHD1 Kısıtlamasının Analizi

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...