Flow cytometry was performed on a LSRFortessa Flow Cytometer (BD Biosciences) and the data were analyzed using FlowJo software. The following antibodies and regents were used: CD8 PB (clone RPA-T8) was from BD Biosciences; CD45RA PE-Cy7 (clone H2100), CCR7 APC (clone 3D12), CD38 PE-Cy7 (clone HIT2) were from eBioscience; CD3 PE/Dazzle594 (clone UCHT1), CD25 PE (clone BC96), HLA-DR ECD (clone L243), T-bet PE (clone 4B10) were from BioLegend; p24 FITC (clone KC57) was from Beckman; Live/Dead™ blue dye (Invitrogen) was used for distinguishing live and dead cells. Measurements of cytokines in cell culture medium were performed with BD™ Cytometric Bead Array Th1/2/17 kit according to the manufacturer’s protocol.
Live dead blue dye
Manufactured by Thermo Fisher Scientific
The Live/Dead™ blue dye is a fluorescent dye used to assess cell viability. It stains dead cells with a blue fluorescence, providing a visual indication of cell health and integrity.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 pb clone rpa t8, by BD (1 mentions)
P24 fitc clone kc57, by Beckman Coulter (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Anti cd8 amcyan, by BD (1 mentions)
Anti ccr7 fitc, by R&D Systems (1 mentions)
Anti pd 1 percpcy5, by BD (1 mentions)
Anti ctla4 pe cy7, by BioLegend (1 mentions)
Anti cd244 fitc, by BioLegend (1 mentions)
Anti cd45ra af700, by BioLegend (1 mentions)
Anti cd3 apc cy7, by BioLegend (1 mentions)
Anti cd160 pe, by BioLegend (1 mentions)
Hla dr percp, by BD (1 mentions)
Cd14 fitc, by BD (1 mentions)
Cd11c apc, by BD (1 mentions)
Cd80 pe, by BD (1 mentions)
Cd86 fitc, by BD (1 mentions)
Cd40 pe, by BD (1 mentions)
Cd45 amcyan, by BD (1 mentions)
Facscanto 2 apparatus, by BD (1 mentions)
Cd83 apc, by BD (1 mentions)
4 protocols using live dead blue dye
1
Comprehensive Flow Cytometry for Immune Cell Analysis
2
Detailed Immunophenotyping of T Cell Subsets
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Immunophenotyping was undertaken following APC-conjugated HLA class I tetramer staining of PBMCs at 37°C for 15 min. Details of the tetramers used can be found in Supplementary Table 5 . Tetramers were conjugated to APC and a true tetramer response was verified through the lack of background staining by gating all CD3+ T cells, against CD8+ T cells and using a tetramer negative control. Surface staining with the following antibodies was then performed: live/dead blue dye (Invitrogen), anti-CD8 Amcyan (BD Biosciences), anti-CD3 APC-Cy7 (Biolegend), anti-PD-1 PercpCy5.5 (BD Biosciences), anti-CTLA4 PE-Cy7, anti-CD244 FITC, and anti-CD160 PE (Biolegend) before washing and flow cytometric analysis. Memory subset analysis utilized the same panel but included anti-CCR7 FITC (R&D systems) and anti-CD45RA AF700 (Biolegend) and omitted anti-CTLA4, anti-CD244, and anti-CD160. Example flow plots can be found in (Figure S1 ).
3
Characterization of Monocyte Subsets by Flow Cytometry
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To characterize monocyte subsets, freshly purified PBMCs were analyzed by six-color flow cytometry on FACS LSRII apparatus. The gating strategy was based on a previous report [15 (link)]. Monocytes were subdivided into three major subsets: classical CD14++CD16−, intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes. The following anti-human mAbs were used: CD45-Amcyan (BD Biosciences), HLA-DR-PerCP (BD Biosciences), CD19-ECD (Beckman Coulter), CD14-QDot655 (Invitrogen), CD16-APC-H7 (Beckman Coulter, Villepinte, France). The Live/Dead blue Dye (Invitrogen) was used to exclude dead cells.
Samples of the purified monocytes used to generate MD-DCs and of the resulting MD-DCs were routinely stained with the following anti-human mAbs: CD14-FITC, CD11c-APC, CD40-PE, HLA-I-FITC, HLA-DR-PerCP, CD80-PE, CD83-APC and CD86-FITC (all from BD Bioscience) and analyzed by flow cytometry on FACS canto II apparatus (BD Biosciences).
Samples of the purified monocytes used to generate MD-DCs and of the resulting MD-DCs were routinely stained with the following anti-human mAbs: CD14-FITC, CD11c-APC, CD40-PE, HLA-I-FITC, HLA-DR-PerCP, CD80-PE, CD83-APC and CD86-FITC (all from BD Bioscience) and analyzed by flow cytometry on FACS canto II apparatus (BD Biosciences).
4
Multiparametric Flow Cytometry of Bat Immune Cells
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The following antibodies (Ab) were used for FACS analyses: goat polyclonal anti-bat IgG (Novus Biologicals, NB7237) detected using an anti-goat IgG secondary antibody (TermoFisher), mouse IgG1 anti-human CD3-FITC (clone CD3-12, Abcam), chicken IgY anti-CADM1 (Necl2, SynCAM1, clone 3E1, MBL) detected using a donkey F(ab)’2 anti-chicken IgY-AlexaFluor647 (Jackson Immunoresearch), rat IgG2b anti-CD44-APC/eFluor780 (clone IM7, eBioscience), rat IgG2b anti-CD11b-Brilliant Violet711 (ITGAM, clone M1/70, BD Biosciences), mouse IgG1 anti-CD172a (SIRPα, clone DH59B, KingFischer Biotech Inc) detected using a polyclonal goat Ig anti-mouse IgG1-PE/Cy7 (batch Poly4053, Biolegend), and rat IgG2a anti-mouse MHCII-FITC (I-A/I-E, clone 2G9, BD Biosciences). Cells (1–5.106 cells/tube) were washed and incubated with Live/Dead blue dye (30 min, 4 °C; Invitrogen/Life Technologies) in PBS. Then, 5% heat-inactivated FBS was added (15 min, 4 °C; Sigma Aldrich). Cells were labelled with antibodies, then washed and stained with secondary reagents. For CD3 intracellular staining, following secondary reagents staining, cells were fixed, permeabilized and stained with the anti-CD3 antibody using the BD Cytofix/Cytoperm kit (BD Biosciences) following manufacturer’s instructions. The samples were acquired using a FACS FORTESSA (BD Biosciences). Analyses were carried out using FlowJo V10 (Tree Star Inc).
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