Exercise 3.4 - Low Speed Separaton of Cells

LEVEL II

Materials

• Sepracell-MN tubes 11
• Clinical centrifuge with fixed angle rotor
• Phase contrast microscope
• Whole Blood 12
• Phosphate Buffered Saline (PBS) + 0.1% (w/v) Bovine Serum
• Albumin (BSA)

Procedure

1. Collect at least 5.0 ml of blood into a Vacutainer containing EDTA as an anticoagulant.

2. Add 5.0 ml of EDTA anti-coagulated whole blood to a Sepracell-MN tube. Mix gently by inversion.

3. Centrifuge at 2,000 xg for 10 minutes at room temperature with a fixed angle rotor. 13
   Decelerate the centrifuge slowly (do not use a brake).

4. Keeping the Sepracell-MN tube upright, insert a Seprapette into the tube and push until the MNC band (mononuclear cells) is displaced into the Seprapette. Do not collect more than 2.5 ml into the Seprapette. After the centrifugation of whole blood, there should be an opalescent compact band just below the meniscus of the tube. This bands contains the mononuclear cells (lymphocytes and monocytes) and platelets. Below this band is the plasma, while the erythrocytes and polymorphonuclear cells will form a dark band at the bottom of the tube.

5. Transfer the contents within the Seprapette by inverting it into a 15 ml conical centrifuge tube. Rinse the inside of the Seprapette with 2.5 ml of PBS and transfer the washing to the conical centrifuge tube.

6. Wash the mononuclear cells in the conical tube by adding 5 ml of PBS containing 0.1% BSA for a total volume of 10 ml and mix by inversion. Centrifuge at 300 xg for 10 minutes at room temperature.

7. Resuspend the mononuclear cells in 5.0 ml of PBS. Make a simple wet mount of the suspended cells and examine the purity of the separation using a phase contrast microscope.

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