Tissue Preparation
Fresh normal leukocytes (WBCs) were obtained by ammonium chloride lysis of peripheral whole blood from healthy donors. Leukocytes from bone marrow were obtained by ammonium chloride lysis of a bone marrow aspirate (BMA). Cell lines were provided by Drs. Lydia McMorrow and Grzegorz Gorski. The cell lines included; a bladder epithelial cell line (T24), a rhabdomyosarcoma cell line (A673), and a Wilms tumor line (SK-NEP-1). Cell lines were grown in high glucose D-MEM medium (Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum, 2mM L-glutamine, and each ml of medium contained 100 U of Penicillin G sodium, 100 U Streptomycin sulfate, and 0.25 µg Fungizone.
Fixation of Cell Suspensions
The 10% formalin solution, buffered in monobasic sodium phosphate (4g/L) and dibasic sodium phosphate (6.5g/L) was obtained from Curtin Matheson Scientific, Inc. (Houston, TX) (Cat. #245-684). Aliquots of approximately 1x106 cells, of each type studied, were fixed by centrifuging (400 x g) the cells, removing the supernatant, and resuspending the cell pellet in 4 ml of the above 10% buffered formalin by vortexing. The cells were incubated in the 10% formalin at room temperature for 1 to 24 hours.
Treatment of Cell Suspensions
The fresh and fixed cells were washed twice in 4 ml of Dulbecco's PBS (Gibco Laboratories, Grand Island, NY), centrifuged (400 x g), and the supernatant removed. Each sample, containing approximately 1x106 of the fixed cells, was then resuspended in 4 ml of one of the following solutions: (1) deionized water, (2) Dulbecco's PBS, (3) 10% methanol in water, (4) 1-12N HCl, (5) 1-12N H2SO4, (6) 1-12N NaOH, (7) 0.5 M 2-mercaptoethanol (2-ME) with 2 M guanidine hydrochloride. The cells were vortexed and then incubated either on ice (4°C), at room temperature (25°C), or in a water bath at temperatures ranging from 37°C to 80°C for various incubation periods up to 4 hours. The cells were then washed twice in 4 mL of Dulbecco's PBS.
DNA Staining
The cells were centrifuged (400 x g) and the supernatant was removed. Each aliquot of cells was then stained by resuspending the pellet in 1ml of PI solution which contained 50 µg of PI, 10 mM Trizma base, 10 mM NaCl, 0.7 U of RNAse, and 1µl of NP-40 and vortexing. The cells were incubated in the PI solution at 4°C for at least one hour before flow cytometric analysis was performed.
Flow Cytometry
Flow cytometry was performed on a Becton-Dickinson FACScan equipped with LYSIS II software. Red-orange fluorescence (FL2) passing through a 585 nm bandpass filter was used to trigger signal processing and the area and width of the linearly-amplified FL2 pulses were collected in list mode for 10,000 events. For each sample, an ungated histogram was generated showing the linear fluorescence intensity for all events collected. This ungated histogram shows, not only the single nuclei, but also the debris and doublets present in each sample. Cell cycle analysis was performed using ModFit (Verity Software House, Topsham, ME). The mean channel of the G0/G1 peak for each sample of fixed cells (untreated and treated) was divided by the mean channel of the G0/G1 peak of the fresh cells from the same donor sample or tissue culture sample. Multiplying this calculation by 100 yields a percentage of fresh cell fluorescence intensity.