Washing the cell isolate🔗

• 1. Estimate the volume of the transferred mononuclear cells. Add at least 3 volumes (~ 6 ml) of balanced salt solution to the mononuclear cells in the centrifuge tube.
• 2. Suspend the cells by gently drawing them in and out of a pipette.
• 3. Centrifuge at 400 to 500 × g for 10 to 15 minutes at 18°C to 20°C.
• 4. Remove the supernatant.
• 5. Re-suspend the mononuclear cells in 6 to 8 ml balanced salt solution.
• 6. Centrifuge at 400 to 500 × g (or 60 to 100 × g for removal of platelets) for 10 min at 18°C to 20°C.
• 7. Remove the supernatant.
• 8. For cell counts, dilute first 1:10 in supplemented RPMI -1640, and then 1:2 with 0.1% trypan blue solution, and count the cells with a Hemocytometer.

MTT Assay—Steps:

• 1. Remove the culture medium from cell monolayers cultured in 96-well plates.
• 2. Reconstitute each well with 0.2 mL supplemented RPMI-1640 containing 1 mg/mL MTT.
• 3. Incubate the cultures for 2–4 hours at 37°C.
• 4. Remove the supernatants from the wells.
• 5. Add 0.2 mL per well of a lysing reagent containing 0.04 N HCl in isopropanol.
• 6. Mix the content of the wells thoroughly.
• 7. Read the plates in an automated microplate spectrophotometer at 570 and 630 nm as reference

References:

Mossman, T. (1983). Rapid colorimetric assay for cellular growth and survival. Journal of Immunological Methods, 65(1-2), 55-63. Sieuweters, A. M., Klijn, J. G., Peters, H. A., & Foekens, J. A. (1995; Nov. 33). MTT tetrazolium salt assay scrutinized. Eur J Clin Chem Clin Biochem, 11, 813-823.

Darzynkiewicz, Z., Li, X., & Gong, J. (1994). Assays of cell viability: discrimination of cells dying by apoptosis, Methods in Cell Biology, 41, 15-38.