4
V79 COLONY FORMING ASSAY with 10% labeling of 131IdU
Experiment Name : 131IdU toxicity (cluster, 10% labeling) Exp. # : Investigator: Date:
Seeded T225 flask with 3 x 106 cells on / / 02 by .
Observation of flask under the microscope after 3 days of incubation period: Cells are
1) ~ % confluent 2) look healthy 3) medium clear
Serum/Lot #s:
1. Set the rocker-roller at 370C incubator with 5% CO2, set the Coulter Counter, wash cells (from one 225 cm2 flasks, seeded with 3 x 106 cells 3 days before) with PBS-PS, trypsinize cells with 2 ml trypsin 3 min at 370C, resuspend in 7 ml MEMB, pool, pass five times through 5 cc syringe with 21 gauge needle, perform cell count by transferring 100 ml in Coulter cup containing 20 ml Isotone II (Coulter balanced electrolyte solution).
Perform cell count in triplicates.
Background: manometer = 500 ml, Bkg. Counts = Av. =
Cells: manometer = 100 ml, Counts Av. =
i.e., Av. x 400 x 5 = x 106 cells / ml
Seed a new flask (P ) with 3 x 106 cells + 30 ml MEMA on / /02 @ hrs.
i.e., 3 x 106 cells = ml
x 106 cells/ml
2. Dilute to A) 400,000 and B) 3,600,000 cells/ml in MEMB
A) Want 11 ml at 0.4 x 106 cells / ml = 4.4 x 106 cells
i.e., 4.4 x 106 cells = ml cells + ml MEMB
x 106 cells / ml
Recount cell count to confirm the above calc. by transferring 100 ml in coulter cup containing 20 ml Isotone II
Cells: manometer = 500 ml, Counts = Av. =
i.e., Av. x 400 = x 106 cells / ml.
B) Want 11 ml at 3.6 x 106 cells / ml = 39.6 x 106 cells
i.e., 39.6 x 106 cells = ml cells + ml MEMB
x 106 cells / ml
Recount cell count to confirm the above calc. by transferring 100 ml in coulter cup containing 20 ml Isotone II
Cells: manometer = 100 ml, Counts = Av. =
i.e., Av. x 400 x 5 = x 106 cells / ml.
3. Transfer 1 ml (400,000 cells) of cell suspension into ten 14 ml tubes and vortex (Falcon plastic test tube labeled 1-10, 17x100 mm) labeled 1-10 both on cap and wall
4. Transfer 1 ml (3,600,000) of cell suspension into ten 14 ml tubes and vortex (Falcon plastic test tube, 17x100 mm) labeled 1U-10U both on cap and wall (U is for unlabeled).
5. Keep the tubes in the roller for 3-4 h at 37°C, 5% CO2 Date/Time:
6. Prepare MEMB containing radioactivity in hood
µl 131IdU (Stock : µCi/µl on ) + ml MEMB
Manufacturer: Lot #: Calibration:
µCi/µl on @ hrs.
on @ hrs.
So conc on @ hrs =
7. After 3-4 h, add 1 ml MEMB to 1U-10U. To the other tubes 1-10, add MEMB with or without radioactivity according to Table below.
Table I.
Tube # / 131IdUµCi/ml / Cells in MEMB
(ml) / MEMB
(ml) / MEMB+
131IdU
[24 µCi/ml]
(ml)
1 / 0 / 1.0 / 1.0 / 0
2 / 0 / 1.0 / 1.0 / 0
3 / 1 / 1.0 / 0.917 / 0.083
4 / 2 / 1.0 / 0.833 / 0.167
5 / 3 / 1.0 / 0.75 / 0.25
6 / 4 / 1.0 / 0.667 / 0.333
7 / 6 / 1.0 / 0.50 / 0.50
8 / 8 / 1.0 / 0.333 / 0.667
9 / 10 / 1.0 / 0.167 / 0.833
10 / 12 / 1.0 / 0 / 1.0
Total / 5.167 / 3.833
7. Return test tubes to roller for 12-14 h. Date/Time:
8. Next day, while test tubes are in roller label 10 gamma-tubes (13 X 100 mm borosilicate glass disposable culture tubes.)
9. After ~12-14 h incubation period, remove all tubes and centrifuge at 2000 rpm at 4°C for 10 min (precooled centrifuge). Date/Time:
10. Remove buckets from centrifuge and carefully remove 150 µl of supernatant from tubes containing radioactivity and place in pre-labeled gamma-tubes. (continue step 27 while the tubes are spinning @ steps 12, 14, 16)
11. Decant supernatant, click tubes, vortex, resuspend in 10 ml wash MEMA
12. Centrifuge tubes for 10 min at 2000 rpm, 4°C
13. Decant supernatant, click tubes, vortex, resuspend in 10 ml wash MEMA
14. Centrifuge tubes for 10 min at 2000 rpm, 4°C
15. Decant supernatant, click tubes, vortex, resuspend in 10 ml of wash MEMA
16. Centrifuge tubes for 10 min at 2000 rpm, 4°C
17. Labeled tubes: (1-10) (0.4 M cell tubes) Decant supernatant, click tubes, vortex, resuspend cells in 5 ml MEMA (regular med.). Syringe 5X with 21G, 5 cc. Transfer 100 µl to Coulter cup w/ 20 ml Isotone II and count.
Unlabelled tubes: (1U-10U) (3.6 M cell tubes): Decant supernatant, click tubes, vortex, resuspend cells in 5 ml MEMA (regular med). 1U – 10U are syringed individually in 14ml Falcon plastic tubes with 5 cc, 21G, 5X and pool all the unlabelled cells into 50 ml (30 x 115 mm Falcon polypropylene conical tube). Transfer 100 µl to Coulter cup w/ 20 ml Isotone II and count.
Table II.
Tube / Coulter1 /
Coulter
2 / Coulter3 / Average / Cell Conc
Cells / ml / Volume (ml)
for 3.6 x 106
1U-10U
Tube / Coulter
1 / Coulter
2 / Coulter
3 / Average / Cell Conc
Cells / ml / Volume (ml)
for 4 x 105
1
2
3
4
5
6
7
8
9
10
18. Transfer 3,600,000 unlabeled cells and 400,000 labeled cells to a new tube.
21. Centrifuge tubes for 10 min at 2000 rpm, 4oC.
22. Decant supernatant completely, click tubes, vortex.
23. Transfer the cell suspension in polypropylene microcentrifuge tubes with attached caps (Helena Plastics, 400 µl) using 200 µl pipette tip.
24. Again add 200 µl ice cold MEMA, resuspend and transfer the cell suspensions in the same polypropylene microcentrifuge tubes (Total volume ~400 µl)
25. Centrifuge tubes for 5 min at 1000 rpm, 4°C
26. Transfer tubes at 10.5°C for 72 h. Date/Time:
27. Transfer 10 µl supernatant in three sets of 7 ml scintillation vials and add 6 ml liquid scintillation cocktail (Ecoscint) from 150 ul supernatant removed earlier (@step 10) and count them for radioactivity. Date/Time:
28. After 72 h, carefully remove the supernatant from the top by using Pasteur pipette, transfer this to ten 14 ml tubes (Falcon plastic test tube, 17x100 mm, labeled 1-10 both on cap and wall) containing 10 ml wash MEMA and resuspend pellet in 200 ml wash MEMA and voretex and transfer the contents to the same ten 14 ml tubes. Date/Time:
29.1 Again add 200 ml wash MEMA in microcentrifuge tubes, resuspend and transfer the cell suspensions in 14 ml tubes by using the same Pasteur pipette.
29.2 Again add 200 ml wash MEMA in microcentrifuge tubes, resuspend and transfer the cell suspensions in 14 ml tubes by using the same Pasteur pipette.
30. Centrifuge the tubes for 10 min at 2000 rpm, 4°C (precooled centrifuge)
31. Labeling and preparation of dilution tubes and colony dishes.
- load 60 tissue culture (petri) dishes with 4 ml MEMA
- load 40 sterile tubes with 4.5 ml MEMA and label them 1.2, 1.3, 1.4, 1.5; 2.2, 2.3, 2.4, 2.5; X.2, X.3, X.4, X.5 etc.
32. Decant supernatant, click tubes, vortex, resuspend in 10 ml wash MEMA
33. Centrifuge tubes for 10 min at 2000 rpm, 4°C.
34. Decant supernatant, click tubes, vortex, resuspend in 10 ml wash MEMA
35. Centrifuge tubes for 10 min at 2000 rpm, 4°C.
36. Decant supernatant, click tubes, vortex, resuspend in 2 ml wash MEMA, pass five times through 5 cc syringe with 21 gauge needle.
37. Determine cell concentration by transferring 100 µl to Coulter cup. (Table III).
38. Vortex tube, transfer 0.5 ml into dilution tube X.5, vortex tube X.5, transfer 0.5 ml into dilution tube X.4, vortex tube X.4 and transfer 0.5 ml to tube X.3, vortex tube X.3 and transfer 0.5 ml to tube X.2 and vortex. Keep tubes on ice.
39. Transfer 1 ml from dilution tubes into dishes labeled X.2, X.3, X.4 (in triplicate). Only X.2 should be seeded for control T-tubes.
40. Incubate tissue culture dishes for 1 week Date/Time
41.1 Vortex and transfer 200 µl of cell suspension (in triplicate) to 12x75 mm polypropylene tubes and label them as C1, C2, C3 etc.
41.2 Centrifuge the remaining cell suspension for 10 min at 2000 rpm, 4°C and transfer carefully only the supernatant of 100 µl (in triplicate) to 12x75 mm polypropylene tubes and label them as M1, M2, M3 etc.
41.3 Collect wipe test tubes for 200 and 1000 ml pipetters, LH3, centrifuse, coulter counter, etc..
42. Count tubes (41.1, 41.2, 41.3) for radioactivity. Date/Time:
43. After 1 week, wash colonies 3 times with normal (1X) saline, and 2 times with methanol. Stain colonies with 0.05% crystal violet
44. Count colonies. (Table IV).
Tube / Coulter 1 / Coulter 2 / Coulter 3 / Average / Cell Conc.1
2
3
4
5
6
7
8
9
10
Table. III.
Tube (X) / Colony CountsDilution
(X.2) / Dilution
(X.3) / Dilution
(X.4) / Dilution
(X.5)
1
2
3
4
5
6
7
8
9
10
Table IV.