Method: Culturing Peripheral Blood Lymphocytes

Rosalie Veile

Principle:

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

Time required:

72 to 96 hours.

Special Reagents:

1. McCoy 5A (TissueSupport Center)
2. Fetal Bovine Serum (Tissue Support Center)
3. L-Glutamine, 200 mM, 100X, (Tissue Support Center)
4. Phytohemmaglutinin (Gibco, Cat. NO. 670-0576 AD)
5. Gentamicin Reagent Solution (50mg/mL),liquid (Gibco, Cat. No. 600-5750 AD)
6. Vacutainer, green top, sodium heparin (Becton Dickinson; Reorder No. 6483)
7. Heparin, sodium salt 300 USP (Sigma, Stock No. 210-6)

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 one or two 7 ml sterile venipuncture tubes coatedwith 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 pipet 10 ml McCoy 5A growth medium to each labeled tube. Add 9-12 drops of whole blood to each, using a sterile 5 3/4" pasteur pipet. Cap the centrifuge tubes tightly and mix gently by inverting tubes. Loosen the caps on the 15 ml tubes and incubate at 37 degrees C in a centrifuge rack positioned at a 45 degree angle for approximately 72 hours.
• Days 2-3
1. Mix the blood by inverting the tubes twice daily during the 3 day incubation period.

   Note: 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.

Solutions:

• McCoy 5A growth medium:

  McCoy 5A	1000.0 ml
  Phytohemagglutinin	10.0 ml
  L-Glutamine	10.0 ml
  Fetal Bovine Serum	100.0 ml
  Gentamicin	1.2 ml
  	1121.2 ml

  Filter sterilize with 0.22 µm cellulose acetate membrane; store medium at 4 degrees C for up to 2 weeks, or freeze medium in 50 ml centrifuge tubes at -80 degrees C for up to 1 year.

References:

Dr. P. Monteleone, Cardinal Glennon Children's Hospital, St. Louis, Mo.