I tried different manners to do the MTT (Cat# 4890-25-K, 2500 Tests) test in SGHPL-4 cells, but I had some troubleshooting.
I incubated 10, 20, 30, 40 and 50 uL of MTT in Hans-F10 medium (with 10% of FBS) for 2, 4, 6, 8, 12 and 24 hours. After that, I put 100 uL of SDS and incubated for 4, 6, and 12 hours both in 37°C or room temperature. Also, I tried to incubate the MTT in PBS for 4 hours, but it did not work.
Did you have any experience with this test/cell?
How can I fix this problem?
Is there another way to perform this test?
No experience with this cell line but many others. Cells grown in 96-well plate in 0.1 mL medium/well. Include a 'no cell' control. Add 20 uL/well of 5 mg/mL MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in PBS (stored in aliquots at -20 C) and incubate 1-4 hrs. Microscopic examination should show dye accumulated in cells. Aspirate off medium and add 100 uL/well DMSO, mix ~15 min on an orbital shaker and read plate at 570nm. Getting the cell density right is usually the biggest challenge. Since the medium plus reagent is removed prior to solubilization in DMSO, it's prudent to visually examine the cells micrscopically beforehand to determine if cell loss will occur with medium aspiration. If the cells are not well attached to the well, then I'd use the MTS [ 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] protocol. Good luck!
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