Peripheral blood mononuclear cells (PBMCs) isolated from healthy or HIV positive individual, are needed in the resting or stimulated condition, for initiation of various virological assays. The method used for separation of PBMCs from blood is as follows. 

Materials required :

1. RPMI 1640 Medium w/L Glutamine
2. Ficoll-hypaque
3. Haemocytometer, cover slips
4. Microscope

Reagents prepared :

1. Washing medium: 2% RPMI
2. Freezing Medium: FBS with 10% Dimethyl Sulfoxide

Procedure:

1. Label sterile, 15 ml tubes with Sample Identification No (NARI Number/ Peripheral No.)
2. In a Laminar flow safety cabinet, load 9ml of blood on 3ml of Ficoll- hypaque using a sterile pipette (Blood to Ficoll ratio should be 3:1). Make sure that the interphase between Ficoll-hypaque (at the bottom) and the blood (on the top) is not disturbed. Take all precautions to prevent contamination of the sample.
3. Centrifuge at x 400 g for 30 minutes at Room Temperature with keeping the acceleration-deceleration at it’s lowest.
4. You will find 4 distinct layrs: RBC layer at the bottom, Ficoll layer, Buffy coat (Cloudy) Layer and plasma as supernatant on the top.
5. Collect the supernatant plasma carefully and distribute in the labeled vials (If Plasma is required), which can be stored at -70^oC.
6. Collect the buffy coat layer (PBMC layer) with minimum amount of Ficoll and transfer it to another labeled 15 ml sterile tube.
7. Add about 10 ml of washing medium and centrifuge at x 225 g for10 minutes.
8. Break the pellet and resuspend in washing medium and repeat step No.7 two times.
9. In the meantime, label cryovials for storing PBMCs using following format and keep the labeled vial in the refrigerator for pre-cooling.
10. PBMC
    Sample ID (Peripheral No.)
    Cell count
    Date of collection
    
11. After second wash, resuspend the pellet in 10ml of RPMI (plain or washing medium) and take the cell count by using trypan blue (0.4% in PBS) in a Neubauer’s counting chamber. Count only the unstained cells. Avoid counting of dead (stained) cells.


12. Dilute 20ml PBMC suspension with 20ml trypan blue and count the cells in Nbaueeur’s chamber. If the number of PBMCs in the suspension is too high, dilute it as 1:10 with washing medium.]

Total PBMC in the suspension = PBMC in all four squares x 10⁴x dilution factor x volume
4

Eg:-     =  88  x  10⁴  x  2  x  10 = 4.4 x 10⁶
               4

13. Centrifuge at x 225g for 10 min to pellet down the cells.
14. Break the PBMC pellet and add pre-cooled Freezing medium to have a final cell concentration of 5 x 10⁶ cells /ml, if cells are to be stored for virus isolation or 3x10⁶ if they are to be stored for PCR.
15. Transfer the cell suspension to the pre-cooled cryovials. To avoid any damage to cells, keep the cryovials immediately in ice.
16. Transfer PBMC vials to Mr Frosty / alcohol bath, (container with isopropyl alcohol which ensures 10^oC cooling per minute) kept at 2-4 oC and transfer it immediately to -70oC freezer.
17. On the next day, transfer PBMC vials to Liquid Nitrogen.