Eccles:MTT Assay (7d) on CB Cells

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MTT Assay kit used was Roche # 11 465 007 001. Each cell line was analysed for proliferation at 33°C with γ-IFN (up-regulation of SV40 large T expression) and 37°C minus γ-IFN (down-regulation of SV40 large T expression). Each day of the assay was carried out in triplicate and each assay was repeated separately three times. One “6 well” sized petri dish (10 x 35mm Falcon dish) of cells was enough to seed two seven day MTT assays of triplicate wells (at 33°C and 37°C). Assay was seeded at 2000cells/well.

MTT Assay (7d) on CB Cells.

Collagen coat required wells at least 2h prior to setting up MTT assay. Refer to (Collagen Coating of Cell Culture Vessels) for protocol.

• Harvest cells as usual by trypsinisation.

• Pellet cells @ 250g, 5min

• Aspirate trypsin/media and resuspend cells in appropriate volume of media (1ml).

• Count cells using haemocytometre to determine number of cells/ml

• Want 20cells/μl x 8000μl. Make dilution of cells to achieve this in 50ml tube.

• Pipette 4ml media into reagent reservoir and using multi-channel pipettor pipette 100μl of media only into media only control wells.

• Empty reagent reservoir and mix 20cells/μl dilution thoroughly and pipette into reagent reservoir.

• Using multi-channel pipettor seed 100μl of 20cells/μl dilution into each well of 37°C assay

• Add appropriate volume of γ-IFN to remaining cells in reservoir (measure how many mls are left and add this many μls of γ-IFN (1x104units/ml)).

• Mix remaining cell dilution (with γ-IFN) thoroughly and using multi-channel pipettor, pipette 100μl into each well of the 33°C with γ-IFN assay.

• Incubate at appropriate temperature.

• Each day just prior to being time to add MTT reagent 1 change the media on all of the wells (aspirate off existing media and replace with another 100μl of media using reagent reservoir and multi-channel pipettor*).

• After 24h from placing experiment in incubator add 10μl of MTT reagent 1 to day 1 triplicates and place back in incubator.

• After 4h add 100μl of MTT reagent 2 to day 1 triplicates and place back in incubator.

• The following morning transfer day 1 well contents to a new 96 well plate and read at 570nm.

• Repeat the above process for day 2 through to day 7.