Rpmi hepes modified

Each sample was thawed quickly in a 37°C water bath by swirling the cyrovial around in the water. Just as the sample thawed, it was transferred using sterile technique to 10 ml of RPMI- HEPES modified (Sigma- Aldrich,R5886) containing 10% FBS, 1% of 200 mM L-glutamine (Gibco, 25030–081) and 1% of 10,000 U/ml penicillin and 10,000 μg/ml streptomycin (Gibco, 15140–122), referred to here on as complete media. The sample was centrifuged at 230xg at RT, media was aspirated, the cell pellet was resuspended in 5 ml complete media, this wash step was repeated and the remaining cell pellet was resuspended in 3 ml complete media. Samples were counted on the Nexcelom Cellometer Auto 2000 Cell Viability Counter using acridine orange and propidium iodide (AO/PI; Nexcelom Bioscience, CS2-0106) to discern live HPBMC from red blood cells (RBC) and dead HPBMC. Cells were brought up to a concentration of 2x10⁶ cell/ml in complete media. HPBMC were assayed for TCP and cytokine production as outlined below.
The preparation, cryopreservation, and shipping of samples in gas phase nitrogen (-180°C) are all critical steps, as well as the thawing and testing of samples to ensure that all sample had greater than 80% viability at the time of testing.

Samples were thawed based on the procedures previously described by Lauer et al. [34 (link)]. Assays consisted of batches of approximately 20 samples. Samples were thawed quickly and washed three times in complete RPMI [RPMI-HEPES modified (Sigma- Aldrich, R5886), with 10% FBS, 1% of 200 mM L-glutamine (Gibco, 25030–081) and 1% of 10,000 U/ml penicillin and 10,000 μg/ml streptomycin (Pen/Strep; Gibco, 15140–122)], referred to here on as cRPMI, then resuspended for counting. Cells were counted on the Nexcelom Cellometer Auto 2000 Cell Viability Counter using acridine orange and propidium iodide (AO/PI; Nexcelom Bioscience, CS2-0106) in accordance with manufacturer’s directions. Samples with viability greater than 80% were used for analysis. Cells were aliquoted for either cell surface marker (CSM) staining or plated in a sterile petri dish for intracellular marker (ICM) staining. ICM samples were placed in a 37°C, humidified, 5% CO₂ incubator overnight to “rest”.