Cd8 percpcy5.5 clone sk1

After 72 h of co-culture, non-adherent cells were transferred to a new plate and 5 μg/mL of Brefeldin A was added. Four-hours later, plates were centrifuged for 3 min at 500 × g and supernatants were removed. Cells were prepared for labeling with cell surface marker monoclonal antibodies (mAb) or conjugated intracellular cytokine mAb as recommended by BD Biosciences. The following mAb conjugates were used: CD3-APCCy7 (clone SK7, BD Biosciences), CD4-PE Texas red (clone S3.5, Caltag/Invitrogen), CD8-PerCPCy5.5 (clone SK1, BD Biosciences), IFNγ-FITC (clone 25723.11, BD Biosciences), MIP-1β-PE (clone D21-1351, BD Biosciences), CD107a-APC (clone H4A3, BD Biosciences), TNF-Brilliant violet 421 (clone MAb11, BioLegend), IL17A-Alexa F700 (clone N49-653, BD Biosciences). Aqua Viability Dye (Molecular Probes/Invitrogen) was added to distinguish live and dead cells. Cells were acquired using an LSRII flow cytometer (BD Biosciences) with FACSDiva software (BD Biosciences). Results were analyzed using FlowJo software (Tree Star).

In the FASCIA method, diluted whole blood is stimulated for seven days with three mitogens ConA, phytohemagglutinin (PHA), and pokeweed mitogen (PWM) as described in detail before [8 (link)]. Minimal blood volume requirement for the test is 0.5 ml. We used the following mitogens and final concentrations in RPMI media (supplemented with 2 mM L-glutamine, HEPES, 100 IU/ml penicillin, and 100 IU/ml streptomycin): PHA 10 μg/ml, ConA 10 μg/ml, and PWM 5 μg/ml (all mitogens from Sigma-Aldrich, USA). PHA and ConA are primarily T cell mitogens, and PWM is widely used to stimulate B cells. Our laboratory has tested that samples can be stimulated after approximately 27 h storage or transport at room temperature without compromising the results (data not shown).
After the incubation, red cells were lysed with IOTest 3 Lysing Solution (Beckman Coulter, USA) and stained with following directly conjugated antibodies: CD8 PerCP-Cy5.5 (clone SK1), CD19 APC (clone SJ25C1), and BD Simultest CD3-FITC/CD4-PE (clones SK7 and SK3 respectively), all antibodies from BD Biosciences, USA. Flow cytometry data was acquired with FACS Canto equipment (BD Biosciences), and the data analyzed with FACS Diva software (BD Biosciences).