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Specific Procedure
For Experiment 6
HT1080, DLD2, and DU145 Cells
Purpose: To find whether synergy exists between
IP6 and PG490 drug concentration combinations in specific cancer
cell lines.
Problem: Do inositol hexaphosphate (IP6) and triptolide
(PG490) induce apoptosis and prevent cell proliferation in HT1080,
DLD2, and DU145 cells?
Hypothesis: If apoptosis is induced and cell proliferation
is prevent then the concentrations of inositol hexaphosphate and
triptolide tested are effective in combating these cell lines, because
the drugs tested are known to prevent cell proliferation and induce
apoptosis on the cells. Induced apoptosis and prevent cell proliferation
can be recognized in the Crystal Violet stain conducted on them,
where the amount of staining in each cell will represent the amount
of cells present.
Materials:
PG490 (Triplotide, Alexis Biochemicals) (MW 360.4) 1 mg in vial.
Add 1 mL 95% ethanol to vial. Incubate at 37ºC for one hour
to dissolve. (Store at –20ºC).
IP-6 (inositol hexaphoshpate, phytic acid, Sigma Chemicals)
(MW 736.2). Dissolve 0.813 g in 2.2 mL DMEM (Dulbecco’s modified
Eagle’s Medium).
PBS and Tryspin(10x dilute) solutions for splitting cells.
DMEM complete: DMEM with 10% fetal bovine serum (FBS), L-glutamine,
and antibiotics.
Cell titer 96 Aqueous non-radioactive cell proliferation assay
Disposables.
Level 2 biosafety cabinet
Humidified 37ºC incubator with 5% CO2
Pipettors, including multichannel pipettor
Hemocytometer and slides
Trypan Blue Assay
Crystal Violet Stain
Prepare IP6 with NaOH Buffer (Refer to Buffering Procedure)
Cell Lines Used:
DLD2 confluent 100 mm dish
HT1080 confluent 100 mm dish
DU145 50% confluent 100 mm dish
Procedure:
1. Trypsinize DLD2 cells (see Sub-culturing procedure). Resuspend
cells in 5 ml DMEM complete and count on hemocytometer. 294 cells
in one 16-square quadrant = 2.94 x 10e6 cells/ml; x 5ml = 1.5 x
10e7 cells total.
2. Set up 96-well microtiter plate, by adding 50 µl of the
following DLD2 dilution to each well of B through H (50 µl
medium only in A); then incubate plates until drug dilutions are
ready:
a. Refer to Hemocytometer Procedure to calculate cell suspension.
b. 2000 cells/well (0.23 ml cell suspension + 14.77 ml DMEM complete)
3. Trypsinize HT1080 cells (see Sub-culturing procedure) Resuspend
cells in 5 ml DMEM complete and count on hemocytometer. 181 cells
in one 16-square quadrant = 181 x 10e4 = 1.81 x 10e6 cells/ml; x
5ml = 9.05 x 10e6 cells total.
4. Set up 96-well microtiter plate, by adding 50 µl of the
following HT1080 dilution to each well in B through H (add 50 µl
medium only to A); then incubate plates until drug dilutions are
ready:
a. Refer to Hemocytometer Procedure to calculate cell suspension.
b. 2000 cells/well (0.26 ml cell suspension + 14.74 ml DMEM complete)
5. Trypsinize DU145 cells (see Sub-culturing procedure) Resuspend
cells in 5 ml DMEM complete and count on hemocytometer. 14 cells
in one 16-square quadrant = 14 x 10e4 = 1.4 x 10e5 cells/ml; x 5ml
= 7.0 x 10e5 cells total.
6. Set up 96-well microtiter plate, by adding 50 µl of the
following DU145 dilution to each well in B through H (add 50 µl
medium only to A); then incubate plates until drug dilutions are
ready:
a. Refer to Hemocytometer Procedure to calculate cell suspension.
b. 8000 cells/well (15 ml cell suspension + 0 ml DMEM complete)
7. Set up PG490 drug dilution microtiter plate:
a. Add 1 µl PG490 @ 1mg/ml to 20 ml medium (= 50 ng/ml)
b. Add 100 µl medium to each well of columns A to G on dilution
plate.
c. Pipette 200 µl of PG490 @ 50 ng/ml into each well of row
H on dilution plate.
d. Do 1:5 serial dilutions of PG490 by transferring 50 µl
of row H samples to the 100 µl in row G. Repeat, transferring
50µl from G to F etc. up to row C. Row A and B have no drug
added.
8. Set up IP6 drug dilution plate:
a. Add 120 µl medium to each well of columns 1 to 10 on dilution
plate.
b. Add 0.06 ml IP6 @ 0.25 M to 5 ml medium. Add 200 µl @3mM
of this dilution to each well of columns 11 and 12.
c. Do 1:3 serial dilutions of IP6 by transferring 30 µl of
col 11 and 12 samples to the 120 µl in col 9 and 10. Repeat.
Col 1 and 2 have no drug added.
9. Add 25 µl from each well of the PG490 dilution plate to
the corresponding wells on the cell plates.
10. Add 25 µl from each well of the IP6 dilution plate to
the corresponding wells on the cell plates.
11. Incubate plates for 5 days. Observations of the plates are made
on the fifth day.
12. Apply Crystal Violet stain to each plate once the data has been
collected. This is done by dumping out the medium and staining the
cells with the Crystal Violet solution. (Refer to Crystal Violet
Stain Procedure for specific instructions)
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