Vi cell counter

All processes for isolating, freezing and thawing cells, ELISpot and ICS were carried out according to validated standard operating procedures (SOPs) employed by the HIL and associated network of Clinical Research Centers against a framework defined by Good Clinical Laboratory Practice (GCLP) accreditation [9] (link). PBMC were isolated from whole blood samples at HIL using Ficoll gradient centrifugation. Upon isolation, PBMC were counted using a Vi-cell counter (Beckman Coulter) and re-suspended in R10 media (RPMI 1640). At this stage 55% of total PBMCs were frozen in freezing media containing 90% FCS and 10% DMSO. Freezing was carried out in a controlled stepwise manner using a rate control freezer and stored in vapour phase liquid nitrogen. The remaining 45% of PBMC were split to assess antigen specific cytokine secretion profiles by IFNγ ELISpot and ICS. Cryopreserved PBMC were thawed rapidly, washed and re-suspended in 5 mL R20 (R10 with 20% FBS) and incubated overnight in a humidified incubator at 37 °C with 5% CO₂ in air. On the day of assay, cells were counted again (using Vi-cell counter, Beckman Coulter) and re-suspended in R10. Samples with viability of less than 80% following overnight incubation were excluded from analysis.

We determined the cell count and viability of shake flask cultures using Vi-cell counter (Beckman Coulter Inc., Fullerton, CA). The glucose, lactate, glutamine and ammonium concentration was determined using YSI biochemical analyzer-Model 2700 analyzer (Yellow Springs Instruments, USA). We used a multi-sampler vapor pressure osmometer (Wescor Inc., USA) to assess the osmolality of the shake flask culture. To quantify the NAD⁺/NADH ratios in parental CHO cells and engineered PYC2 expressing clones, we used NAD⁺/NADH Assay Colorimetric Kit (Abcam, USA) and followed the same protocol as provided by the supplier. The IgG concentration was measured using high-performance liquid chromatography (HPLC, Waters, USA) Protein-A affinity column (Analytical column). The protein samples were collected at different time intervals to analyze the antibody titer.