AIM: PREPARATION OF PLASMA AND ISOLATION OF LEUKOCYTES

FROM THE GIVEN BLOOD SAMPLE.

Requirements:
1. Anticoagulated blood sample
2. Sterile Pasteur pipette
3. 15 ml sterile centrifuge tubes
4. Sterile PBS (KH2PO4- 0.24 g, NaH2PO4-1.44 g, KCl- 0.2 g, NaCl- 8 g in 1 litre of
water, pH-7.4)
5. Hisep solution (Himedia)

Methods:

1. 4 ml of heparinized blood is taken in 15 ml centrifuge tube and it is diluted with 4

ml of PBS.
2. 4 ml of Hisep solution is taken in a 15 ml centrifuge tube and carefully the diluted
blood is layered on the Hisep solution without disturbing it.
3. This is centrifuged at 1500-2000 rpm for 20 min.
4. After centrifugation, a buffy coat (containing the leukocytes) is formed at the
interface of upper plasma layer and lower RBC.
5. The buffy coat is isolated out very carefully with the help of a micropipette.
6. If plasma layer is transparent, it can be aspirated in a separate tube and stored for
further use. If plasma layer appears to be non-transparent which means it may
have some suspended leukocytes can be purified by spinning at 2500rpm for 10
min which yield clear and transparent plasma and a pellet of leukocytes.
7. The leukocytes are washed in PBS at 1500-1700 rpm for 10 min. Aspirate out the
supernatant.
8. A part of the leukocyte suspension is stored in PBS at -20°C and a part of it is be
washed 2-3 times with culture media and finally suspended in complete media.