PBMC Protocol

Cryopreserved peripheral blood mononuclear cells (PBMC) are a frequent source of specimens for research that includes immunotherapies and responses to vaccines. While some protocols are limited to isolation and freezing phases of the process, others include thawing procedures.

Isolation Procedure and Freezing Procedure

• A commercial compound to promote separation is placed into each 50 mL centrifuge tube and the tubes are centrifuged for thirty seconds. Caps of blood tubes are sprayed with alcohol before opening, then anticoagulant-treated whole blood is then transferred via pipette into the centrifuge tubes. The plasma layer of the the blood is aspirated with a pipette.

Freezing

• The cells from the previous step will then be centrifuged with buffered saline three times. After a cell count, the specimens will be centrifuged then suspended in freezing medium. Specimens in freezing medium are next divided between cryovials in one mL amounts. Vials are stored overnight in a

  -70ºC freezer and moved to long-term storage in liquid nitrogen the next day.

Safe Thawing

• Cryovials are transferred from liquid nitrogen to a 37ºC water bath and specimen is allowed to thaw one to two minutes. A warm transport media is added to each vial. Diluted cell suspension is centrifuged for seven minutes. The number and cell viability are checked, If cells need concentrating, specimens are centrifuged seven more minutes. The sample is diluted to working solution and rested for twelve to eighteen hours before beginning examination in the specified protocol.