Ficoll purification of PBMCs from TRIMA Leukoreduction chambers

Materials and Reagents

• Ficoll-Paque Ficoll® Paque Plus GE Healthcare, 17-1440-02, pack of 6 × 100 mL

• Leukoreduction Chamber from Trima Apheresis Collection (blood center of the pacific) http://www.bloodcenters.org/hospitals-research/products-services/

• 50 ml Falcon tubes

• Cryo Vial

• Thermo Scientific™ Mr. Frosty™ Freezing Container (Thermo Scientific: 5100-0001

• Isopropanol to fill the Mr. Frosty

• PBS

• FBS

• RPMI

• Penn/Strep

Equipment

• Centrifuges

A-Ficoll

1. Wipe exterior of blood pack with EtOH 70, especially the tubing

2. Prepare scissors (clean with EtOH 70)

3. Prepare a Falcon 50 ml tube

4. Transfer blood from TRIMA into the falcon tubes by cutting first the bottom tube then the top let the blood drip

5. Add to PBS to obtain 40 ml

6. Divide the 40 ml into 2 falcon 50 (ie transfer out 20 ml in a fresh tube)

7. Add PBS to 35 ml in both tubes

8. Add 12.5 ml Ficoll to two new 50 ml falcon tubes and Layer blood on top of the Ficoll, being careful to not to disrupt the interface let any stick to the sides.

9. Note: the 12.5 ml of Ficoll can also be layer underneath the diluted blood but be aware of not making bubbles while layering beneath

10. Spin 30 minutes at 2,000 rpm at RT, no brake

11. Remove ~10 ml of Supernatant (ie above the buffy)

12. Slowly pipet out the 5-10 ml of interface, with as little Ficoll as possible, and transfer to a fresh 50ml falcon tube

13. Add 20 ml PBS

14. Pellet, wash twice

15. Count cells: one TRIMA unit gives about 500 million cells

B-Freeze the PBMCs:

1. Freeze PBMCs that are not needed

2. Pellet left over cells

3. Carefully remove the supernatant and resuspend in Freezing Media (90 FBS +10% DMSO) for having 50 million cells per vial of 500ul

4. Transfer the cell solution into cryo vials

5. Place the cryovials in the blue Mr. Freeze container previously filled with appropriate level of isopropanol (~200ml). Transfer the container into a -80˚C freezer and leave overnight

   Note: Alternatively, if Mr. Freeze container are not available, the vials can be placed in the middle of an emptied Styrofoam rack used for 15ml falcon tubes. Another emptied Styrofoam rack is placed on the top is taped with the other one to maintain the vial inside and not open it by mistake when in the freezer. The goal is to have a slow decrease in temperature overnight

6. Transfer the vials into a liquid nitrogen container (log the samples)

C-Thaw the PBMCS:

1. Take vial out and incubate in a water bath at 37˚C (log sample sheet)

2. Transfer the cell suspension in a 15 ml falcon

3. Add slowly 10 ml of RPMI 10% FBS with Penn/Strep

4. Pellet left by centrifuging at 1,500 rpm for 5 min

5. Carefully remove the supernatant and resuspend in fresh R slowly 10 ml of RPMI 10% FBS with Penn/Strep

6. Count and proceed to experiment