Culturing Peripheral Blood Lymphocytes

Principle
Peripheral blood lymphocytes are incubated in a defined culture medium supplemented with serum, phytohemaglutinin, and other additives for the purpose of preparing metaphase chromosomes.


Time Required
72 to 96 hours.


Special Reagents

1. McCoy 5A (Tissue support center)
2. Fetal Bovine Serum (Tissue support center)
3. L-Glutamine, 200 mM, 100X, (Tissue support center)
4. Phytohemaglutinin
5. Gentamicin Reagent Solution (50 mg/mL), liquid
6. Vacutainer, green top, sodium heparin
7. Heparin, sodium salt 300 USP

Safety Considerations
Because we do not test the cell lines or blood samples, always work under the assumption that the cells carry infectious agents, e.g., HIV virus, hepatitis B, etc. Keep the samples isolated, work only in the biological safety hoods, and always wear gloves. Autoclave all waste materials.

Procedure
Day 1

1. Collect blood by venipuncture in 1 or 2 7-mL sterile venipuncture tubes coated with sodium heparin. Rotate the tubes to prevent clotting. Label tubes with individual’s name, date, and time of drawing the specimen.
   Store at room temperature (never place in refrigerator or on ice).
2. Label 8–10 15-mL centrifuge tubes, then pipette 10 mL McCoy 5A growth medium to each labeled tube. Add 9–12 drops of whole blood to each, using a sterile 5 ¾" Pasteur pipette. Cap the centrifuge tubes tightly and mix gently by inverting tubes. Loosen the caps on the 15-mL tubes and incubate at 37°C in a centrifuge rack positioned at a 45° angle for approximately 72 hours.

Days 2-3

1. Mix the blood by inverting the tubes twice daily during the 3-day incubation period.
   
   If clumps form at the bottom of a culture tube, 5–10 drops of heparin (300 USP) can be added with a 1-cc syringe.

Solution

• McCoy 5A growth medium:
  
  

  McCoy 5A Phytohemaglutinin L-Glutamine Fetal bovine serum Gentamicin

  1000.0 mL 10.0 mL 10.0 mL 100.0 mL 1.2 mL

  
  
• Filter sterilize with 0.22 mm cellulose acetate membrane; store medium at 4°C for up to 2 weeks, or freeze medium in 50-mL centrifuge tubes at –80°C for up to 1 year.
• Phytohemaglutinin, M form, PHA, lyophilized
• Rehydrate with 10 mL sterile double distilled water (ddH₂O). Store at
• 2°C–8°C. After reconstitution, the phytohemaglutinin solution is good for 14 days.
• Heparin, sodium salt 300 USP: Rehydrate with 5 mL of McCoy growth medium. Store at 4°C for up to 2 weeks.