Thomas Wang
October 13, 2002
Long-term project-methods
Meeting 2 Section B
Methods:
Cell lines
Cell lines used were Ind-8 for asymmetric kinetics and Con-3 for control exponential kinetics.
Ind-8 is a cell line with Zinc-controlled p-53 protein expression, which regulates asymmetric
kinetics.
Cell growth and radiation treatment
(I need to add in schematics for the experiment design)
General Cell culture
Cells were thawed from liquid nitrogen storage and incubated initially in plain media
(Dulbecco’s Modified Eagle Medium (DMEM), dialyzed fetal bovine serum (DFBS), and
penicillin streptomycin) in T-75 flasks for 24 hours. Media was changed to puro media (plain
media with 5 ug/ml puromycin) and maintained. Cells were split every 3-4 days using standard
techniques with Hank’s Balanced Salt Solution (HBSS) to wash cells of old media, trypsin to
detach cells from flask, and fresh puro media to return cells to incubation. For experiments cells
were initially plated at a concentration of 2 x 10^4 cells/mL and incubated at 37 C for 24 hours.
Two experiments were conducted and each used 3 control flasks for Con-3, 3 control flasks for
Ind-8, 1 experimental flask for Con-3, and 1 experimental flask for a total of 16 flasks for both
experiments.
Experiment 2
After initial plating and 24 hour incubation of cells, bromodeoxyuridine (BrdU) was added to all
flasks at 20 uM concentration. Cells were allowed to grow for 24 hours and then puro media
was changed to zinc media (puro media with 75 uM zinc chloride) to induce asymmetric cell
kinetics in Ind-8 cells. BrdU was also added to new zinc media at 20 uM concentration again
and Tritiated S-Adenosyl methionine (3H SAM) was added to each of the experimental flasks at
4.04 uCi/ml concentration. After another 24 hours of incubation, cells were harvested.
Experiment 3
After initial plating and 24 hour incubation of cells, BrdU was added to all flasks at 20 uM
concentration. Cells were allowed to grow for 24 hours and then puro media was changed to
zinc media to induce asymmetric cell kinetics in Ind-8 cells. BrdU was also added to new zinc
media at 20 uM concentration. Cells were incubated for another 24 hours and then 3H SAM was
added directly to each of the experimental flasks at 4.04 uCi/ml concentration without changing
media. After another 24 hours of incubation, cells were harvested.
Harvesting cells
Cells were harvested using standard techniques with HBSS and trypsin to wash and detach cells
from flask. Cells were then spun down in conical tubes at 4 C for 5minutes at 1500 RPM to
make pellets. Media was aspirated and then cells were resuspended in cold phosphate buffer
saline (PBS) to wash down the samples. Cells were then spun down again at 4 C for 5 minutes at
1500 RPM to give final pellet. For harvesting, the 3 control flasks of Con-3 and the 3 control
flasks for Ind-8 were combined to result in 4 samples for each experiment: control Con-3 and
Ind-8 and Experimental Con-3 and Ind-8. Cell counts were also taken with a coulter counter for
later analysis of asymmetric kinetics.
DNA extraction, quantification, and scintillation counting
DNA extraction
Cell pellets were incubated with 0.5 ml cell lysis buffer (need to find the contents) and 200 ug/ml
proteinase K for 3 hours at 50C to lyse all cells and denature proteins. Cell pellets were then
extracted with 0.5 ml of phenol:choloroform by centrifuging sample for 1 minute and then
transferring the aqueous, DNA-containing layer to a fresh tube. The extraction process was then
repeated with .5 ml chloroform to purify solution of cell debris and proteins. The DNA solution
was incubated again with 5 ul (100 ug/ml) RNAse A for 1 hour at 37C and then the
phenol:chloroform extraction was repeated two more times and the choloroform extraction once
more in order to remove RNA and further purify the DNA sample. Samples were then stored
overnight in 1 ml of 100% cold ethanol and 50 ul NaOAc to assist in precipitation of the DNA
from solution. After overnight storage, samples were spun down for 30 minutes at max speed at
– 20C. After ethanol was decanted, samples were allowed to sit for 5 minutes before
resuspension in 1 ml 70% cold ethanol. Samples were spun down for 15 minutes again at max
speed at – 20C. Ethanol was decanted and samples dried for 30 minutes before reconstitution of
DNA in 100 uls of TE buffer (10 mM Tris-Cl, pH 8.0 and 1mM EDTA, pH 8.0)
DNA quantification
Total DNA amounts in each sample were quantified using picogreen analysis following
manufacturer’s directions. 5 ul of sample was added to 95 ul of TE and 100 ul of picogreen dye
(diluted 1/200 in TE) for each well in a 96 well microplate. Samples were mixed on a tilt plate
for 5 minutes at high speed and then analyzed with a fluorimeter. The standard curve was
constructed using Con-3 DNA with no radiation.
Scintiallation counting
Five ul of each sample was added to 5 ml of scintillation fluid in a glass scintillation counting
vial. Samples were loaded into scintillation counter and counts per minute (cpm) were
determined.
Gradient analysis of DNA samples
Cesium chloride gradient
Cesium chloride solution was prepared with a final refractive index of 1.401 and then filtered
through a .25 u filter to remove particulates. Eight mls of CsCl was added to centrifuge tubes
and then between 10 ug and 40 ug of DNA samples (in TE pH 8.0 solution) were added. The
volume added of each sample varied based on the DNA concentration determined earlier. TE
buffer was also used to balance tubes to +/- 0.01 g. Tubes were sealed and tube number, sample
id and rotor slot were recorded for each sample. Samples were then spun at 40,000 rpm at 25 C
for 20 hours. Samples were removed from the rotor and dripped into microplate at a rate of 3
drips/well (~150-200 ul/fraction).
DNA quantification and scintillation counting
The DNA amount in each fraction of each sample was determined using picogreen analysis as
described above. Similarly, cpm in each sample was determined using above protocol with the
modification of using 80 ul of each fraction sample for counting.