[cell biology work] MTT_assay

[Seung Jae Jeong]

MTT assay

This procedure is for adherent cells in 96 well plates,

if larger plates are used then adjust volumes accordingly.

 

1. Make a solution of 5 mg/ml MTT dissolved in PBS and filter sterilise.

2. 5 hours before the end of the incubation add 20 μl of MTT solution from step one to each well containing cells.

3. Incubate the plate in a CO2 incubator at 37ºC for 5 hours.

4. Remove media with needle and syringe.

5. Add 200ul of DMSO to each well and pipette up and down to dissolve crystals.

6. Put plate into the 37ºC incubator for 5 minutes.

7. Transfer to plate reader and measure absorbance at 550nm.

 

Brief protocol

1. Plate cells (104-106 cells)in 96-well (flat form).

2. Incubate for appropriate time.

3. Add 25μl of MTT soln. to each well.

4. Incubate for further 4hr.

5. Decant gently supernatant.

6. Add 100μl of DMSO, and resuspend the pellet for 30 min during shaking.

7. Measure OD in 540nm.

 

자세한 방법

96 well plate 에다 (culture된 Hela cell을 희석하여, 약 200ul씩 multipipette으로 첨가) Hela Cell을 한 80% 키운 다음(하룻저녁)에, media를 걷어내고, sample이 treat된 media를 다시 넣어준다. (이때 media는 serum free)

(sample이 treat된 media는 새로운 96 well plate에 미리 준비해 둔다. 최고농도에 10mg/ml sample을 10.24ul/400ul media에 넣고 multipipette으로 serial dilution)

12시간 정도 incubation 한 다음에 MTT solution (5mg/ml)을 20μl 씩 넣어주고 2시간 – 4시간 정도 더 incubation 한다.

Media를 다시 제거하고, DMSO 원액 200ul를 다시 분주한 다음, 570nm에서 흡광도를 측정한다.

 

 

 

 

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