Can I use 3H-Thymidine on adhesive cell for cell proliferation assay - (Aug/23/2006 )
Can I use 3H-Thymidine on adhesive cell for cell proliferation assay?
do u mean adherent cell line?? if so yes...let me know if u need the protocol.
Can I use 3H-Thymidine on adhesive cell for cell proliferation assay?
do u mean adherent cell line?? if so yes...let me know if u need the protocol.
Thank you so much. I do need the protocol
ok this is what I do with human breast cancer cells and it works well.
1. Plate 0.5 million cells /well in six well plates
2. Treat it accordign to you rprotocol for predetrmined time -points.
3. Add 4 ul thymidine (4 uCi) to each well one hour prior to your TP. ie for 24 h u will thymidine at 23 h and for 48 h it will be at 47 h and so on.
4. Put your plate back into the incubator.
5. At the end of your time-point place plates on ice and pull off media. Ideal to work near the sink and you dont need sterile conditions after this point.
6. Discard media in appropriate containers (as it will be radioactive).
7. Wash wells with 2 ml of 1X PBS (ice cold). Discard appropriately.
8. Add 2 ml 5% TCA to each well and incubate on ice for 5 mins.
9. Wash with 5% TCA twice more.
10. Add 1 ml of 1N NaOH to each well and incubate at 37C (i just use a warm room or oven) for 10 mins.
11. Meanwhie prepare ur scintillation vials.
12. Add the end of 10 mins add 1 ml of 1N HCl.
13. Collect the 2 ml in scintillation vials and add 4ml scintillation fluid. ( my vial capacity is 7 ml and hence these quantities)
14. Cap and shake well and count in a scintillation machine.
good luck and always do triplicates.
ps: if ur cells are not coming off well increase the NaOH incubation time.
Thank you so much for your help.
1. Plate 0.5 million cells /well in six well plates
2. Treat it accordign to you rprotocol for predetrmined time -points.
3. Add 4 ul thymidine (4 uCi) to each well one hour prior to your TP. ie for 24 h u will thymidine at 23 h and for 48 h it will be at 47 h and so on.
4. Put your plate back into the incubator.
5. At the end of your time-point place plates on ice and pull off media. Ideal to work near the sink and you dont need sterile conditions after this point.
6. Discard media in appropriate containers (as it will be radioactive).
7. Wash wells with 2 ml of 1X PBS (ice cold). Discard appropriately.
8. Add 2 ml 5% TCA to each well and incubate on ice for 5 mins.
9. Wash with 5% TCA twice more.
10. Add 1 ml of 1N NaOH to each well and incubate at 37C (i just use a warm room or oven) for 10 mins.
11. Meanwhie prepare ur scintillation vials.
12. Add the end of 10 mins add 1 ml of 1N HCl.
13. Collect the 2 ml in scintillation vials and add 4ml scintillation fluid. ( my vial capacity is 7 ml and hence these quantities)
14. Cap and shake well and count in a scintillation machine.
good luck and always do triplicates.
ps: if ur cells are not coming off well increase the NaOH incubation time.
1. Plate 0.5 million cells /well in six well plates
2. Treat it accordign to you rprotocol for predetrmined time -points.
3. Add 4 ul thymidine (4 uCi) to each well one hour prior to your TP. ie for 24 h u will thymidine at 23 h and for 48 h it will be at 47 h and so on.
4. Put your plate back into the incubator.
5. At the end of your time-point place plates on ice and pull off media. Ideal to work near the sink and you dont need sterile conditions after this point.
6. Discard media in appropriate containers (as it will be radioactive).
7. Wash wells with 2 ml of 1X PBS (ice cold). Discard appropriately.
8. Add 2 ml 5% TCA to each well and incubate on ice for 5 mins.
9. Wash with 5% TCA twice more.
10. Add 1 ml of 1N NaOH to each well and incubate at 37C (i just use a warm room or oven) for 10 mins.
11. Meanwhie prepare ur scintillation vials.
12. Add the end of 10 mins add 1 ml of 1N HCl.
13. Collect the 2 ml in scintillation vials and add 4ml scintillation fluid. ( my vial capacity is 7 ml and hence these quantities)
14. Cap and shake well and count in a scintillation machine.
good luck and always do triplicates.
ps: if ur cells are not coming off well increase the NaOH incubation time.
If you have a automatic cell harvester, you can save a lot of time, especially if you are using 96 well plate
1) freeze the plate after the time point
2) thaw the plate
3)harvest the cell onto glass filter.
4) add scintillation fluid
5) read the filter using TOP COUNT machine.
Hope this may help.
If you have a automatic cell harvester, you can save a lot of time, especially if you are using 96 well plate
1) freeze the plate after the time point
2) thaw the plate
3)harvest the cell onto glass filter.
4) read the filter using TOP COUNT machine.
Hope this may help.
[/quote]
Hello, Minnie:
I am a little confused by your method. I always use trypsin and then harvest cell. If freeze and thaw, will it break the cell.
Plus, for this experiment, I have the problem that the values from triplicate are not consistent and the the values are jumping too much , not good for fitting. I am using 96-well plates. Do you have any good idea for it.
Thanks
I am a little confused by your method. I always use trypsin and then harvest cell. If freeze and thaw, will it break the cell.
Plus, for this experiment, I have the problem that the values from triplicate are not consistent and the the values are jumping too much , not good for fitting. I am using 96-well plates. Do you have any good idea for it.
Thanks
The adherence cells do not break, they detached from the plates...only freeze and thaw once.
The use of typsin to lift the adherence cells is very messy and dangerous because of radioactive isotope. It is OK if you have only less than 10 wells to trypsin...not practical in 96 well plates.
When seeding the cells,
1) mix the cells by inversion gently in a 50ml tubes.
2) pour the mixture into a 100mm culture dish
3) use a good mutichannel pipette to aliquot quickly
4) swirl the culture dish after seeding half the plate
5) aliquot the rest.
To minimize the variation among triplicates, try to aliquot the cell into the triplicate well at the same time.
I normally divide the plate into four columns (each contains 3x8wells),then use a multichannel pipette with 6 tips and aliquot the cells mixture into half the row (say A1-A6, A8-A12, B1-B6, B7-B12).
Since the cells do settle a little over time in the culture dish, this method can ensure the same cell density in each triplicates.
I hope this may help.
, all right!, Thanks a lot, Minnie Mouse. 