Alexa fluor 488 anti human cd19

Alexa Fluor® 700 anti-human CD3, (clone: HIT3a), FITC-anti human TCRαβ (clone: IP26), FITC-anti-human TCRγδ (clone: B1), APC/Cy7 Anti-Human CD127 (I-7Ra), (clone: A019D5), PE anti-human CD161, (clone: HP-3G10), Brilliant Violet 421TM anti-human CD117 (c-kit), (clone 104D2), PE/Cy7 anti-human CD294 (CRTH2), (clone: BM16), APC anti-human CD336 (NKp44), (clone: 325110), Alexa Fluor® 488 anti-human CD19, (clone: HIB19), FITC anti-human CD94, (clone:DX22), FITC anti-human CD1a, (clone: HI149), FITC anti-human CD11c, (clone: 3.9), FITC anti-human CD123, (clone: 6H6), anti-human CD303 (BDCA-2), (clone:201A), FITC anti-human CD14, (clone: 63D3), FITC anti-human FcεRIα, (clone: NP4D6), FITC anti-human CD34,(clone: 561), APC/Cy7 anti-human IFN-γ, (clone: 4S.B3) all from BioLegend. Anti-Human CD363 (S1PR1) eFluor® 660, (clone: SW4GYPP, ThermoFisher), Mouse IgG1 K Isotype Control eFluor® 660, (clone: P3.6.2.8.1, ThermoFisher). Anti-mouse CD3) APC, (clone: 17A2), anti-mouse NK1.1 Alexa Fluor® 647, (clone: PK136), anti-mouse B220 APC/Cy7 or FITC, (clone:RA3-6B2), anti-mouse CD45-Percp Cy5.5, (clone:30-F11), anti-mouse CD90.2 PE Cy7, (clone:30-H12), anti-mouse CD11b APC, (clone:M1/70), anti-mouse GATA3 PE (Clone: 16E10A23), anti-mouse Rorγt (Clone: 2F7-2).

The PBMCs from the 16 to 18-h incubations were harvested and stained on ice in staining media (PBS with 2% FBS and 0.02% sodium Azide) with the following antibodies: Fc block (anti-CD32), Brilliant Violet 421™ anti-human CD40 Ligand, Brilliant Violet 605 anti-human CD4, Alexa Fluor® 488 anti-human CD19, PE-Cy7 anti-human CD69, and Alexa Fluor® 647 anti-human CD86 (BioLegend). The cells were washed twice by centrifugation at 350×g and stained with the fixable viability dye zombie NIR (BioLegend) in PBS at 1:1000 dilution for 30 min. on ice and then washed again in cell-staining media. Legendplex ultracomp compensation beads (BioLegend) were stained with 1/10th concentration of the above antibodies. ArC™ Amine compensation beads (Thermofisher) were stained with zombie NIR fixable viability dye. The stained cells and beads were analyzed on a 4-laser Cytoflex flow cytometer (Beckman Coulter). Compensation and cell analysis was performed on FlowJo software (Tree Stars). T and B cells were identified as CD4⁺ or CD19⁺ positive respectively after gating on single, live lymphocytes and further analyzed for the expression of activation markers, CD69, CD86, and CD40L. Fluorescence minus one (FMO) controls negative/negative gates.