Radioactive Thymidine Assay
PART ONE
Things you’ll need before starting:
- Treated cells in 24-well plates, about 80% confluent
- At least an hour of time (15-30 min + 45 min wait time)
- Thymidine (3H) (found in radioactive fridge in hard plastic box in blue container)
- Normal Ins-1 media
- 10% TCA (kept in big clear bottle in the radioactive fridge)
- 1x PBS (kept in big clear bottle in the radioactive fridge)
- 0.3 M NaOH (kept in big clear bottle in the radioactive fridge)
- **Anything that comes into contact with radioactivity (pipette tips, plates, etc.) needs to go in radioactive labelled bags or waste bucket.
PROCEDURE:
1. Make up 3H media in the designated bottle by adding 0.25 uL thymidine per 1 mL of normal media ( *for islets use 1 ul thymidine /ml media). You will need enough for 500 uL per well (for 3x 24 well plates use 10 uL thymidine into 40 mL media).
2. Dump media from 24 well plates down radioactive waste drain.
3. Replace the 24 well plates' media with 500 uL 3H media per well.
4. Incubate for 15 minutes at 37*C. During this time, mark the 3H book inventory with the amount of thymidine used and the disposal method.
5. Dump 3H media down radioactive waste drain.
6. Wash each well with 250 uL of 1x PBS three times. Dump down radioactive waste drain after each wash.
7. After dumping the last wash, add 500 uL of cold 10% TCA to each well and incubate in radioactivity fridge for 30 minutes.
8. Dump TCA.
9. Add 250 uL of cold 0.3 M NaOH to each well.
10. Seal plate with parafilm and freeze. Can leave plate frozen overnight or continue assay after at least 30 minutes in the freezer.
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PART TWO
Things you’ll need before starting:
- At least 1.5 to 2 hours to load the samples (the scintillation counter takes about 7 hours for 72 vials)
- Verify nobody else will be using the scintillation counter at the same time as you. You can also stake your claim via sticky note.
PROCEDURE:
1. Thaw plate from Radioactive Thymidine Assay at room temperature.
2. Label each scintillation tube. Use the blue racks to line up the tubes.
3. Add 50 uL from each well into each corresponding tube.
4. Add one pump (about 4 mL) of scintillation fluid per scintillation tube. Be careful not to get fluid on the outside of the tubes.
5. Put tubes in scintillation counter rack (it reads back to front). In first rack put the “1” flag as the start code. In the first empty rack place the “stop” flag.
6. Press run. It will take about 5 min per vial (3 standard vials also in machine). When done results will print out on printer below machine. May have to press "okay" on printer to get it to go.
7. Run the Radioactive Thymidine BCA.
Radioactive Thymidine BCA
Things you’ll need before starting:
- Sufficient BCA standards
- Sufficient BCA Protein Assay Reagents A and B (kept around radioactive desk)
- F-bottom 96 well plates (non-cell culture)
- Some form of lid to prevent evaporation (i.e. plate lid, parafilm, aluminum foil)
PROCEDURE:
1. Set up/label your plates. You will need one set of standards per plate. You will run each of your samples in triplicate.
- Example: For one 24 well plate of samples, you will have one 96-well plate with 9 standards and 72 wells of samples (24*3).
2. Aliquot sufficient BCA reagents for 50 parts A to 1 part B (51:1)
Make enough for 150 uL in each well (round up so you have a little extra). For one 24 well plate, you will need to fill 81 wells total.
Calculations if you round up to 83:
83*150uL = 12,450 uL total
12,450 uL/51 = 244.12 uL of B
- 244.12 uL*50 = 12,206 uL of A
3. Reserve the first 9 wells for standards A-I, then add 10 uL of each sample to the plate.
4. Add 10 uL of each standard to reserved wells.
5. Mix together A and B. Add 150 uL of BCA Reagent mix to each well.
6. Incubate for 30 min to 2 hours (until color is sufficiently developed).
7. Read plate on plate reader at 562 nm (May be saved as BCA protocol). Save results to an excel file.