I isolate lymphocytes from leukapheresis filters. I try to proliferate the lymphocytes with PHA-P, but after MTT Assay the control is the same or higher. I use 100.000 cells in a 96wells plate and 5μg/ml PHA. The MTT is performed the third day of incubation (10 μl Mtt/well). I incubate 4 hours with mtt, centrifuge the cells, because they are not adherent and I dissolve the precipitate with 200μl of DMSO. After 15min I read the plate in Elisa reader. Any suggestions?
Dear Maria
Using 100,000 cells per well in 96w plate in some cases are too much. Although the exact number of cells in MTT assay depends on your cell growth kinetic but as you keep cells for 72h many of them may be died due to shortage of nutrition or space which affect your results, I suggest to use 50,000 cells per well instead.
Good Luck
Hello Maria
The cell density of 100,000 cells per well in a 96-well plate is causing abnormal results. I suggest you first finalize your seeding density for MTT assay by running an experiment with a range of seeding densities in a 96-well plate for 72 hours. I would recommend you to use 5000 to 10000 cells per100ul per well in a 96-well plate keeping in mind the 72 hour incubation period.
I would prefer you to use XTT assay instead of MTT assay because XTT produces water soluble formazan dye and so there is no need of the solubilization step. Minimizing the number of steps in your assay will minimize errors.
Good Luck.
Dear Maria!
Please You look at following article:
Evaluation of a new lymphocyte proliferation assay based on cyclic voltammetry; an alternative method
https://www.nature.com/articles/s41598-019-41171-8
Lymphocyte proliferation studies
Duplicate wells in each plates were set-up for concurrent determination of proliferation by colorimetric MTT (tetrazolium) and cyclic voltammetry (CV) assays. One hundred microliters of diluted cell suspension (2 × 106 cells/mL) in complete RPMI (RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin) were dispersed and incubated in 96-well microtiter plate. Cells were seeded in 10 wells per sample for each test (mitogen added) and control negative experiment. The mitogen phytohemagglutinin-L (PHA-L; Sigma) at the final working concentrations of 5, 10 and 20 µg/mL was added to test wells. In addition, cell-free media containing complete RPMI were run in parallel to test and control groups. The inoculated plates were incubated at 37 °C in a 5% CO2 incubator and checked for growth after 4, 24, 48 and 72 h using both colorimetric and CV assays. Each assay is further evaluated for sensitivity to change in cell number and different concentrations of mitogen. Six different concentrations of cells in complete RPMI media ranging from 3 × 106 to 1.2 × 104 cells/mL (3 × 105 − 1.2 × 103 cells/well) were tested by both colorimetric MTT and CV assays. All the experiments reported here were repeated four times.
Colorimetric MTT assay
Colorimetric lymphocyte proliferation was evaluated using MTT (3-(4, 5-dimethyl thiazol-2-yl) 2, 5-diphenyl tetrazolium bromide) method. After cell culture incubation (4, 24, 48 and 72 h), 10 μl MTT (dissolved in RPMI-1640, 5 mg/mL) was added to well, and plates were further incubated at 37 °C for 4 h. Finally, the purple formazan crystals were dissolved by adding 100 μl acid-isopropanol (0.04 N HCI in isopropanol) into each well. Absorbance was then measured at 550 nm against reference wavelength of 630 nm and stimulation index (SI) was determined. The SI is expressed with average OD value in the test group divided by average OD value in negative controls.
Thank you very much for the answers. Regarding the number of cells, i have also performed BrdU colorimetric assay with 20.000 cells/well following the given protocol, but i have the same problem
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