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.