VPB 321: Animal Biotechnology (2+1)

ISOLATION OF LYMPHOCYTE

Aim

• Isolation of lymphocytes from cattle blood for culturing in vitro.

Material required

• Heparinized syringes, Eagles Media with Phytohaemagglutining, culture flasks, colcemid, Incubator, Conical centrifuge and tubes.

Procedure

• Collect 5 ml of venous blood from a cattle in a sterile syringe containing 0.5 ml of sodium heparin (1000 units/ml)
• Bend a clean, covered 18 gauge needle to a 45⁰ angle and place on the syringe and allow the syringe to stand on a platform with needle pointing up, plunger down for 90-120 minutes at room temperature.
• During this time, the erythocytes settle by gravity, leaving app. 4ml of leucocytes-rich plasma on the top and a white bufy coat of leucocytes in the middle.
• Carefully expel the leukocyte-rich plasma and buffy coat from the syringe into a sterile tissue culture flask containing 8 ml of Eagle’s Media supplemented with 0.1 ml of phytohaemagglutinin (PHA).
• Incubate the culture for 66-72 hrs at 37⁰C in an incubator. Gently agitate the cultures once or twice daily during the incubation period.
• Add 0.1 ml of colcemid (10 micrograms/ml) to the culture flasks and incubate for an additional 2 hours.
• Transfer the colcemid-treated cells to a 15 ml centrifuge tubes and centrifuge at 1000 rpm for 5 minutes.
• Aspirate and discard all but 0.5 ml of the supernatant. Gently tap the bottom of the centrifuge tube to resuspend the cells in the remaining 0.5 ml of culture media.

Questions

• Write the purpose of using Heparin for collecting blood samples?
• What is the action of Phytohaemagglutinin?
• What is the use of Eagle’s Medium in leukocyte culturing?
• What is the purpose of adding colcemid into the leukocyte culture?
• Why leucocytes collected for chromosome analysis?