PBMCs isolation from CPT™ tube

Mix the blood sample immediately prior to centrifugation by gently inverting the tube 8 to 10 times.

Centrifuge the CPT tube at 1500rcf,0h 0m 0s (Relative Centrifugal Force) in a horizontal rotor (swing-out head) for 0h 30m 0s at 20°C (Speed change of accel/decel: Soft).

Using a Pasteur pipette, aspirate approximately half of the plasma without disturbing the PBMC cell layer.

Collect cell layer by pouring and transferring cell layer to a 50 mL size conical centrifuge tube with cap.

Spin down the collected mixture at 300rcf,0h 0m 0s for 0h 15m 0s at 20°C.

Using a 5-mL serological pipette, gently resuspend the cell pellet with 3mL of ACK lysing buffer and incubate for 0h 3m 0s at Room temperature.

First wash: Add wash buffer to bring volume to 50mL. Cap tube. Mix cells by inverting tube 5 times.

Centrifuge at 300rcf,0h 0m 0s (accel/decel: Soft) for 0h 15m 0s at 20°C.

Aspirate as much supernatant as possible without disturbing cell pellet.

Second wash: Add wash buffer to bring volume to 20mL. Cap tube. Mix cells by inverting tube 5 times.

Centrifuge at 300rcf,0h 0m 0s (accel/decel: Soft) for 0h 15m 0s at 20°C.

Aspirate as much supernatant as possible without disturbing cell pellet.

Re-suspend cell pellet in an appropriate volume of wash buffer to bring to a concentration of ~1×10⁶ cells/mL for counting.

Cell counting:

Mix 10µL of cell suspension with 10µL of trypan blue.

Apply 10µL of the mixture to a counting slide.

Count the cells using an automated cell counter within 0h 5m 0s (concentration tends to range from 5×10⁴ to 1×10⁷ cells/mL).

Centrifuge the remaining suspension at 300rcf,0h 0m 0s (accel/decel: Soft) for 0h 10m 0s at 20°C.

Aspirate as much supernatant as possible without disturbing cell pellet.

Resuspend cell pellet in 1mL of cold CryoStor CS10 in cryotubes and aliquot into two cryotubes per sample (0.5 mL X 2).

Store the cryotubes into CoolCell LX Freezing Container in a -80°C freezer 0h 10m 0s before permanent storage in liquid nitrogen.