Intracellular cytokine staining (ICS) assays were done using whole blood, TCL (6 hours) or PBMC (overnight), stimulated with JEV peptides (3–10 μg/ml), peptide pools (3 μg/ml), JEV infected cell lysate, or approximately 103.4 to 104.4 plaque forming units (PFU) of JEV SA14-14-2 in the presence of 10 μg/ml brefeldin A during the stimulation. Following stimulation (and red cell lysis in the case of assays using whole blood), cells were stained with near infrared viability dye (Invitrogen) at room temperature in the dark for 20 minutes, fixed with 2% formaldehyde at room temperature for 20 minutes, and cryopreserved at -80°C in PBS/1% bovine serum albumin/10% DMSO. Later, cells were incubated in FACS perm/wash buffer (BD) at room temperature for 20–30 minutes followed by staining in perm/wash buffer for 30 minutes at 4°C. Antibody clones used for anti-CD3, CD4 and CD8 were as above. Antibodies used for TCL ICS were: anti-CD3-FITC, anti-CD4-PerCP-Cy5.5, anti CD8-APC, anti-IFNγ-PE or PE-Cy7 (clone 4S.B3), anti-IL2-PE (clone 5344.111) and anti-TNFα-PE-Cy7 (clone MAb11). Antibodies used for ex-vivo ICS were: anti-CD3-AmCyan, anti-CD4-PerCP-Cy5.5, anti-CD8-Horizon v450, Anti-CD14-APC-Cy7 (clone MφP9), anti-IFNγ-PE-Cy7, anti-TNFα-APC, anti-IL2-PE and anti-MIP-1β-FITC (clone D21-1351). MIP-1β was from R&D systems, all other antibodies were from BD.
Facs perm wash buffer
Manufactured by BD
The FACS perm/wash buffer is a laboratory reagent used in flow cytometry applications. It is designed to facilitate the preparation and processing of cell samples for flow cytometric analysis.
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2 protocols using facs perm wash buffer
1
Intracellular Cytokine Staining for JEV Peptides
2
Multiparametric Flow Cytometry Immunophenotyping
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Cells were stained for 30 min at 4°C in PBS containing 0.01% NaN3 and 0.5% BSA with directly labeled antibodies against: CXCR3 Alexa fluor488, CCR5 PE, CCR4 PerCP‐Cy5.5, CCR7 PE‐Cy7, CCR6 Alexa647, CD4 APC‐H7, CD3 V500, CD69 PerCP, CD45 V500 (all from BD Biosciences); CD3 FITC (Sanquin); CD4 PE‐Cy7, CD45 RA eFluor450, and CD45RO PE (all from eBioscience Inc., San Diego, CA). For intracellular cytokine staining, we used IL‐10 PE‐Cy7 (BioLegend, San Diego, CA, USA) and the Th1/Th2/Th17 kit from BD Biosciences. For intracellular staining, after cell surface staining, cells were washed and fixed with FACS Cytofix fixation buffer (BD Biosciences). Following permeabilization (FACS Perm/Wash buffer [BD Biosciences]) cells were stained with markers as indicated. Cells were analyzed on a FACS Canto II (BD Biosciences). CS&T beads were run daily and the same machine with dedicated cytometer configuration was used for the measurements of the samples throughout the study with regular control runs to check compensation settings. Data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
Data were plotted as frequency of positive cells or as the gMFI for cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest were normalized to the gMFI of the cytokine negative population.
Data were plotted as frequency of positive cells or as the gMFI for cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest were normalized to the gMFI of the cytokine negative population.
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