Procedure: Isolation of Mononuclear Cells (MNC)🔗

Human Mononuclear cells can be separated from heparinized blood samples or from the buffy coat of centrifuged blood sample with sodium citrate as an anticoagulant after removal of the erythrocytes and the plasma. The residual fraction (25–40 ml) contains more than 90% of the leukocytes, 10% of the erythrocytes, and 5% of the plasma.

• 1. Dilute buffy coats 1:4 (v/v) or heparinized blood samples 1:2 (v/v) with Dulbecco's Phosphate buffered saline (PBS) without Ca2+ and Mg2+.
• 2. To a 10 ml centrifuge tube add 2 ml of defibrinated or anticoagulant treated blood and an equal volume of balanced salt solution (final volume 4 ml).
• 3. Mix the blood and buffer by inverting the tube several times or by drawing the mixture in and out of a pipette.

Procedure for isolation of mononuclear cells

• 1. Invert the Ficoll-Paque media bottle several times to ensure thorough mixing.
• 2. Using aseptic techniques, add Ficoll-Paque media (3 ml) to the centrifuge tube.
• 3. Carefully layer the diluted blood sample (4 ml) onto the Ficoll-Paque media solution.
• 4. Centrifuge at 400 g for 30 to 40 minutes at 18ºC to 20°C (brake should be turned off).
• 5. Draw off the upper layer containing plasma and platelets using a sterile pipette, leaving the mononuclear cell layer undisturbed at the interface.
• 6. Transfer the layer of mononuclear cells to a sterile centrifuge tube using a sterile pipette.

A Micro-titer Plate after MTT Assay Tetrazolium salts are converted by metabolically active cells to formazan, which can be quantified by measuring absorbance at 570 nm.