TITLE: PBMCs isolation from CPT™ tube
AUTHORS:   Woong-Yang Park,   Jay Shin, Shyam  Prabhakar

[DESCRIPTION]
This protocol details the procedure for collection and isolation of blood samples using CPT tubes.

[BEFORE_START]
The BD Vacutainer® CPT™ Tube (Cat. no.362753) should be at Room temperature (18-25ºC) and properly labeled for patient identification. 
After blood collection, the CPT tube should be stored upright at Room temperature (18-25ºC) until centrifugation. Blood samples should ideally be centrifuged within two hours of blood collection for best results.


[STEPS]
SECTION: PBMCs isolation from CPT™ tube
1. Mix the blood sample immediately prior to centrifugation by gently inverting the tube 8 to 10 times.
SECTION: PBMCs isolation from CPT™ tube
2. Centrifuge the CPT tube at 1500 rcf (Relative Centrifugal Force) in a horizontal rotor (swing-out head) for 30 min at 20 °C (Speed change of accel/decel: Soft).

SECTION: PBMCs isolation from CPT™ tube
3. 
 
Using a Pasteur pipette, aspirate approximately half of the plasma without disturbing the PBMC cell layer.
SECTION: PBMCs isolation from CPT™ tube
4. Collect cell layer by pouring and transferring cell layer to a 50 mL size conical centrifuge tube with cap.
 

SECTION: PBMCs isolation from CPT™ tube
5. Spin down the collected mixture at 300 rcf for 15 min at 20 °C. 
 


SECTION: PBMCs isolation from CPT™ tube
6. Using a 5-mL serological pipette, gently resuspend the cell pellet with 3 mL of ACK lysing buffer and incubate for 3 min at Room temperature.

SECTION: PBMCs isolation from CPT™ tube
7. First wash: Add wash buffer to bring volume to 50 mL. Cap tube. Mix cells by inverting tube 5 times.

SECTION: PBMCs isolation from CPT™ tube
8. Centrifuge at 300 rcf (accel/decel: Soft) for 15 min at 20 °C. 

SECTION: PBMCs isolation from CPT™ tube
9. Aspirate as much supernatant as possible without disturbing cell pellet.
SECTION: PBMCs isolation from CPT™ tube
10. Second wash: Add wash buffer to bring volume to 20 mL. Cap tube. Mix cells by inverting tube 5 times.

SECTION: PBMCs isolation from CPT™ tube
11. Centrifuge at 300 rcf (accel/decel: Soft) for 15 min at 20 °C.
SECTION: PBMCs isolation from CPT™ tube
12. Aspirate as much supernatant as possible without disturbing cell pellet.

SECTION: PBMCs isolation from CPT™ tube
13. Re-suspend cell pellet in an appropriate volume of wash buffer to bring to a concentration of ~1×106 cells/mL for counting.
SECTION: PBMCs isolation from CPT™ tube
14. Cell counting:
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14.1. Mix 10 µL of cell suspension with 10 µL of trypan blue.
SECTION: PBMCs isolation from CPT™ tube
14.2. Apply 10 µL of the mixture to a counting slide.
SECTION: PBMCs isolation from CPT™ tube
14.3. Count the cells using an automated cell counter within 5 min (concentration tends to range from 5×104 to 1×107 cells/mL).

SECTION: PBMCs isolation from CPT™ tube
15. Centrifuge the remaining suspension at 300 rcf (accel/decel: Soft) for 10 min at 20 °C.
SECTION: PBMCs isolation from CPT™ tube
16. Aspirate as much supernatant as possible without disturbing cell pellet.
SECTION: PBMCs isolation from CPT™ tube
17. Resuspend cell pellet in 1 mL of cold CryoStor CS10 in cryotubes and aliquot into two cryotubes per sample (0.5 mL X 2).

SECTION: PBMCs isolation from CPT™ tube
18. Store the cryotubes into CoolCell LX Freezing Container in a -80 °C freezer 10 min before permanent storage in liquid nitrogen.