Research Paper Volume 3, Issue 11 pp 1078—1091

Figure 1. Defining mTOR-dependent CS. (A) Measurement of CS. 80000 HT-p21-9 cells were plated in 0.2 ml medium per well in 96-well plate with 500 nM rapamycin (R) or without rapamycin – control (C). After 4 days, the plate was photographed to record color of media (i.e. pH). The color of medium without cells is shown for comparison n/c (no cells). Then cells from each well were split into larger wells of 6-well plates. Detailed description: medium (together with floating cells) was aspirated and adherent cells were trypsinized in 0.2 ml of trypsin. Equal volume (a 4 μl aliquot of cell culture) or 2% of total adherent (live) cells was plated in 4 ml of fresh medium in 6-well plates. After 7 days, colonies were stained and photographed. A number of colonies is a measure of viability of the stationary culture. (B) Time-dependent (chronological) loss of replicative viability. Cells were plated at initial density of 40000 and 80000 cells per well in 96-well plates without (C) or with rapamycin (R) as shown in panel A. After indicated time, cells were trypzinised and equal volume of adherent cells (2%) were re-plated in 6 well plates and allowed to form colonies for 7 days. (C) Cells from plates shown in panel B were trypsinized and counted to precisely quantify replicative viability. (D) Levels of lactate in conditioned medium. Cells were plated at initial density of 40000 and 80000 cells per well in 96-well plates without (C) or with rapamycin (R) as shown in panel A. After indicated time, concentration of lactate was measured in conditioned media.