Tc20 automated cell counter

PBMCs were quickly thawed in a 37°C water bath, rinsed with culture medium (RPMI 1640 medium supplemented with 15% FBS and 1% Pen/Strep) and then treated with 0.2 U/μL DNase I in 10mL of culture medium at 37°C for 30 min. After DNase I treatment, cells were washed with medium once, filtered with a 35 μm cell strainer and cell viability and concentration were measured with trypan blue on the TC20 Automated Cell Counter. Cell viability was greater than 90% for all samples. Cells were plated at a concentration of 1 × 10⁶ cell/mL, rested at 37°C and 5% CO₂ for 1 h and then treated with the specified concentrations of the following stimulants (or DMSO as a control) for either 1h or 6h:

20 ng/mL Lipopolysaccharide (LPS) (tlrl-3pelps, Invivogen),

50 ng/mL Phorbol 12-myristate 13-acetate (PMA) (P8139, MilliporeSigma) + 250 ng/mL Ionomycin calcium salt (I0634, MilliporeSigma),

20 ng/mL Interferon gamma (IFN-Ɣ) (RP1077, Cell Applications)

For the “Golgi Inhibitor” experiments, cells were incubated for 6 h with GolgiPlug (555029, BD Biosciences) at a 1:1000 dilution plus stimulants at the concentrations indicated above (or GolgiPlug only as a control).
After stimulation, cells were washed twice with ice-cold 1x PBS + 0.1% BSA and cell viability and concentration were measured with trypan blue on the TC20 Automated Cell Counter.