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Protocol

MTT ASSAY
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MTT - powder from Sigma. Store at 4°C. Stock - 5 mg/ml in PBS. Filter sterilize. Store at 4°C in the dark. Make up small amounts and use within a few weeks. If blue crystals precipitate out, refilter. RPMI without PR - commercially available. Isopropanol or Isopropanol/0.04 N HCl
I. Method for Adherent Cells 1. Plate cells in microtiter plates to desired density. 2. Remove media by gently inverting plate. Tap plate on gauze to remove drops of media. 3. Add 100 ml RPMI (without phenol red): 10% MTT stock. 4. Place in incubator for 3-4 hours. 5. Add 100 ml isopropanol to each well. 6. Pipette up and down 2-3 times to dislodge bottom crystals. 7. Place on "Flow" plate shaker for 20 minutes to dissolve crystals. Do not set speed very high. 8. Read in ELISA reader at 570-690 nm within 1 hour after adding isopropanol. II. Method for Non Adherent Cells 1. Plate cells to desired density in microtiter plates. Use 100 ml media per wells. 2. Add 10 ml MTT stock to each well. 3. Incubate 3-4 hours. 4. Add 100 ml isopropanol/HCl (0.4 N) to each well. 5. Pipette up and down 2-3 times to dislodge bottom crystals. 6. Place on shaker for 20 minutes to dissolve crystals. Do not set speed very high. 7. Read in ELISA reader at 570-690 nm as soon as possible after it is finished shaking.
1. There are two advantages of using method I for adherent cells as opposed to the simpler method II. The first is that by using PRMI without phenol red you eliminate the need to acidify the medium with HC1. This increases the sensitivity of the assay approximately 20%. The second is that by changing media you eliminate serum proteins which can precipitate when the isopropanol is added. 2. The MTT in solution is not very stable as mentioned above. 3. The sensitivity of the assay varies between 200 and 3000 cells per well. References Mossman, J. Immuno. Methods 65 (1983):55-63 Denizot, F. & Lang, R.J.Immunol. Methods 89 (1986):271-277

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