2.6 CHO Cell Lysis and Determination of Cell Number
Upon completion of a 72 hr growth cycle, the RBL drive system was disengaged and cells were allowed to settle. Growth media was removed from the culture wells and transferred to labeled 0.5mL tubes. The media/cell supernatant was centrifuged at 1000 rpm for 5 min. to pellet any intact suspended cells remaining in growth media. At this time, a volume of 100 µL Triton X-100 (1%) was added to each well containing the settled cells and gently mixed by pipet to disassociate any large clusters. This plate was left undisturbed for 10 min. to ensure complete lysis. Meanwhile, the supernatant/media was removed from 0.5mL tubes while being careful not to disturb the pellet. The lysed cell solution (100 µL) was transferred to its corresponding 0.5mL tube, vortexed (2-3 pulses), and allowed to incubate for another 5 min. to complete lysis of any residual cells from media suspension. After this period, 0.5mL tubes were centrifuged at 5,000 rpm for 5 min. to spin down any cell debris and leaving soluble protein in supernatant. A volume of 10 µL from the supernatant solution was transferred to a clear 96-well plate for protein analysis. The detergent compatible Bradford protein assay was run according to manufacturer recommended microplate procedures and read at 595 nm using multi-mode plate reader (BioTek Synergy LX). In order to relate soluble protein absorbance values to cell count, lysate prepared from cell populations with known concentrations ranging from 0 to 1 X 10^6 cells/mL was evaluated for protein content. A standard curve of soluble protein to cell count was generated and cell populations are reported as cells/mL.