BD Vacutainer® CPT™ Cell Preparation Tube with Sodium Citrate

Procedure

1. The BD Vacutainer® CPT™ Tube with Sodium Citrate should be at room temperature (18-25° C) and properly labeled for patient identification.
2. Collect blood into the Tube using the standard technique for BD Vacutainer® Brand Blood Collection Tubes (see Venipuncture Technique & Sample Collection section and Prevention of Backflow section).
3. After collection, store Tube upright at room temperature until centrifugation. Blood samples should be centrifuged within two hours of blood collection for best results.
4. Centrifuge Tube/blood sample at room temperature (18-25° C) in a horizontal rotor (swing-out head) for a minimum of 20 minutes at 1500 to 1800 RCF (Relative Centrifugal Force).

   NOTE: Remix the blood sample immediately prior to centrifugation by gently inverting the Tube 8 to 10 times. Also, check to see that the Tube is in the proper centrifuge carrier/adapter.

   WARNING: Excessive centrifuge speed (over 2000 RCF) may cause tube breakage and exposure to blood and possible injury. To calculate the correct centrifuge speed for a given RCF, use the following calculator. Simply enter two of the three values in the calculator and press the Calculate button.

   Radius (cm):
   R.C.F. (gravities):
   Rotating Speed (rpm):

   

5. After centrifugation, mononuclear cells and platelets will be in a whitish layer just under the plasma layer (see above figure). Aspirate approximately half of the plasma without disturbing the cell layer. Collect cell layer with a Pasteur pipette and transfer to a 15 mL size conical centrifuge Tube with cap. Collection of cells immediately following centrifugation will yield best results.
6. An alternative procedure for recovering the separated mononuclear cells is to resuspend the cells into the plasma by inverting the unopened BD Vacutainer® CPT™ Tube gently 5 to 10 times. This is the preferred method for storing or transporting the separated sample for up to 24 hours after centrifugation. To collect the cells, open the BD Vacutainer® CPT™ Tube and pipette the entire contents of the Tube above the gel into a separate vessel.

Suggested Cell Washing Steps:

1. Add PBS to bring volume to 15 mL. Cap Tube. Mix cells by inverting Tube 5 times.
2. Centrifuge for 15 minutes at 300 RCF. Aspirate as much supernatant as possible without disturbing cell pellet.
3. Resuspend cell pellet by gently vortexing or tapping Tube with index finger.
4. Add PBS to bring volume to 10 mL. Cap Tube. Mix cells by inverting Tube 5 times.
5. Centrifuge for 10 minutes at 300 RCF. Aspirate as much supernatant as possible without disturbing cell pellet. Resuspend cell pellet in the desired medium for subsequent procedure.