Immune cell populations identification
anti-PD1-PE-Cy7, anti-CD15-PacificBlue
FLUOR, LYSIS
To decipher the peripheral immune system, we performed five panels to identify and characterize the different lymphocyte and myeloid subpopulations.Antibodies for T cell analysis (first panel): using Beckman Coulter’s custom design service and its dry coating technology, custom tubes containing anti-CD183-FITC (clone G025H7), anti-CD197-PE (clone G043H7), anti-CD196-PE-Cy7 (clone B-R35), anti-CD278-APC (clone ISA-3), anti-CD45RA-Alexa Fluor 700 (clone 2H4LDH11LDB9 (2H4)), anti-HLA-DR-APC-Alexa Fluor 750 (clone Immu-357), anti-CD4-PacificBlue (clone 13B8.2) and anti-CD8-KromeOrange (clone B9.11) were produced. Liquid antibodies were also used: anti-CCR4-PerCP-Cy5.5 (BioLegend, clone L291H4) and anti-CD28-BV605 (BD Biosciences, clone CD28.2).Antibodies for T cell analysis (second panel): using Beckman Coulter’s custom design service and its dry coating technology, custom tubes containing anti-CD183-FITC (clone G025H7), anti-CD197-PE (clone G043H7), anti-CD196-PE-Cy7 (clone B-R35), anti-PD1-APC (clone PD1.3), anti-CD45RA-Alexa Fluor 700 (clone 2H4LDH11LDB9 (2H4)), anti-CD4-PacificBlue (clone 13B8.2) and anti-HLA-DR-KromeOrange (clone Immu-357) were produced. Liquid antibodies were also used: anti-CD80-APC-Alexa Fluor 750 (BD Biosciences, clone L307.4) and anti-CD127-BV605 (BioLegend, clone A019D5).Antibodies for Treg cell analysis: using Beckman Coulter’s custom design service and its dry coating technology, custom tubes containing anti-CD25-PE (clone B1.49.9), anti-CD39-PE-Cy5 (clone BA54), anti-PD1-PE-Cy7 (clone PD1.3), anti-CD278-APC (clone ISA-3), anti-CD45RA-Alexa Fluor 700 (clone 2H4LDH11LDB9 (2H4)), anti-CD4-PacificBlue (clone 13B8.2) and anti-CD8-KromeOrange (clone B9.11) were produced. Liquid antibodies were also used: anti-CCR4-PerCP-Cy5.5 (BioLegend, clone L291H4), anti-CD80-APC-Alexa Fluor 750 (BD Biosciences, clone L307.4) and anti-Tim3-BV605 (BioLegend, clone F38-282).Antibodies for natural killer (NK) cell analysis: using Beckman Coulter’s custom design service and its dry coating technology, custom tubes containing anti-CD159a-PE (clone Z199), anti-PD1-PE-Cy5 (clone PD1.3) anti-CD335-PE-Cy7 (clone BAB281), anti-CD314-APC (clone ON72), anti-CD56-APC-Alexa Fluor 750 (clone N901), anti-CD16-PacBlue (clone 3G8) and anti-CD45-KromeOrange (clone J33) were produced. Liquid antibodies were also used: anti-Tim3-FITC (Miltenyi Biotec, clone REA635), anti-NKG2C-Alexa Fluor 700 (R&D Systems, clone 134591) and anti-CD3-BV605 (BioLegend, clone UCHT1).Antibodies for myeloid cell analysis: multi-color flow cytometry was also performed using Beckman Coulter’s custom design service and its dry coating technology, and custom tubes containing anti-CD33-FITC (clone D3HL60.251), anti-CD39-PE (clone BA54), anti-CD3-Pe-Cy5 (clone UCTH1), anti-CD19-PE-Cy5 (clone J3-119), anti-CD20-PE-Cy5 (clone B9E9), anti-CD56-PE-Cy5 (clone N901), anti-PD-L1-APC (clone PDL1.3.1), anti-HLA-DR-APC-Alexa Fluor 750 (clone Immu-357), anti-CD15-PacificBlue (clone 80H5), anti-CD14-KromeOrange (clone RMO52) and a mortality marker DRAQ7 were produced. The following liquid antibody was added to the custom tubes: anti-CD11b-BV605 (BioLegend, clone ICRF44).Staining protocol: 100 μl of total heparinized blood was added to each DURAClone tube containing liquid antibodies, vortexed immediately for 15 s and incubated for 15 min at room temperature in the dark. Two milliliters of red blood lysis solution (VersaLyse solution, A09777, Beckman Coulter) containing 50 μl of the fixative agent IOTest 3 Fixative Solution (A07800, Beckman Coulter) was added, inverted and incubated for 15 min in the dark. After centrifugation and washing with 3 ml of PBS 1×, cells were resuspended in 150 µl of PBS 1× before acquisition on a Canto II cytometer (BD Biosciences).