[박상규] PBMC isolation using Ficoll-Paque Plus(amersham)

[Anonymous]

PBMC isolation using Ficoll-Paque Plus(amersham)

 

When you use 50ml blood….

 

Materials

50ml conical tube (8ea)

Ficoll : 45ml

Heparin coated blood collection tube: 5개è Do NOT use EGTA tube

RPMI(serum free)-room temperature equilibrated

 

Before you start blood pick up…

Add 7.5ml Ficoll (2.4cm) to each 50 ml tube and equilibrate at RT

 

1.      Pick up blood 10ml(10mlX5 person) into Heparin coated blood collection tube and invert tube carefullyè DO NOT put the blood tube in cold space.

2.      If you pick up the blood from individual persons, pool the blood

3.      Dilute blood with RPMI media 1:1 ratio.

4.      Load carefully 16ml of 1:1 diluted blood to the Ficoll cushion (7.5ml). Be careful Not to interfere the cushion layer.

5.      Centrifuge at 400g for exactly 40 min at 18-20 degree

6.      Draw off the upper layer using a clean Pasteur pipette, leaving the lymphocyte layer undisturbed at the interface. Care should be taken not to disturb the lymphocyte layer. The upper layer of plasma, which is essentially free of cells, could be stored for later use.

7.      Using a clean Pasteur pipette, transfer the lymphocyte layer to a clean 50ml conical tube

8.      Add at least 3 volume of RPMI to the lymphocyte in the test tube and suspend the cells carefully with pasteur pipette

9.      Centrifuge at 100g for exactly 10 min at 18-20 degree

10.   Remove the supernatant

11.   suspend the lymphocytes in 10ml RPMI media by gently swirling

12.   Repeat step 8&9.

13.   The lymphocytes should be now suspended in the proper media (usually RPMI).

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