Protocols

Tritiated thymidiene Uptake assay and MTT assay

Posted on Saturday, October 18, 2003

Description
Tritiated thymidiene Uptake assay and MTT assay
To study if modulation of apoptosis by CD40 ligation and TNFa activation is by induction of proliferation or inhibition of apoptosis, following assays were performed

Procedure
The cells (6 X 104 cells per 200ml per well) were incubated with CD40 antibody/ TNFa for the indicated time points in a 96 well U bottom plate(Nunc, USA). The microwells received 0.5 mCi 3H-TdR (specific activity: 6500 mCi/mmole) 18 hrs, before harvesting the cells on to glass fibre filters using a Titertek cell harvester (Flow Laboratories, Norway). The samples were counted using a liquid scintillation analyzer (LKB,Sweden). The cell viability was also assessed by colorimetric MTT assay essentially as described (Tada et al, 1986). 10 µl of MTT (5 mg/ml) was added to each well 4 hrs before the termination of the assay. The reaction was terminated by adding 100 µl of acidified SDS to each well. The plate was kept overnight at room temperature to allow the formazan crystals to dissolve. The developed color was read in a multiplate reader (Labsystems, UK) at 540 nm with a reference wavelength of 690 nm.

Recipes
1. Tritiated thymidiene (3H-TdR): 3H-TdR (specific activity 6-9 Ci/mM) was obtained in 50% ethanol from the Isotope division , Bhabha Atomic Research Center, Mumbai. The stock solution of 1 mCi/ml was diluted in sterile saline to give a final concentration of 200 µCi/ml, aliquoted and stored at 4°C. For further use, each aliquot was further diluted to 50 µCi/ml and 0.5 µCi was added to each microtitre well.
2. Scintillation Fluid:
PPO (2,5, Diphenyloxazole, Sigma, USA): 7.0 g
POPOP (1,4-bis [5-phenyloxazolyl] benzene, Sigma, USA): 0.5 g
Toluene AR grade (SRL, India): 1000 ml.
This was stored at room temperature.
3. MTT (3-[4,5-dimethylthiazole-2-yl] 2,5,-diphenyl tetrazolium bromide): 500 mg of MTT (Sigma, USA) was dissolved in 100 ml of normal saline to obtain a concentration of 5 mg/ml. It was sterilized by millipore filtration and stored in dark bottle at 4°C. It was used at a final concentration of 0.5 mg/ml.
4. Acidified SDS solution: 15% of sodium dodecyl sulphate (SRL,Mumbai) was prepared in 0.15 mM Hydrochloric acid. This solution was stored at room temperature and used to terminate the MTT assay.


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