IsoFlux Human PBMC Preparation, Freezing and Thawing (With CPT Tube)

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Intended Use

This protocol may be used to prepare peripheral blood mononuclear cells (PBMC) from human blood collected in BD Vacutainer® CPT tube, freeze, and thaw the PBMC for IsoFlux rare cell isolation. Note that this approach may lead to varied CTC counts compared to fresh sample enrichment. 

Materials

  • BD Vacutainer® CPT™ Mononuclear Cell Preparation Tube Sodium Citrate (8mL, Cat. 362761)
  • PBS (phosphate buffered saline, Ca2+ Mg2+ -free)
  • RPMI 1640 media
  • CTL-Wash™ Supplement (Immunospot, Cat. CTLW-010)
  • Fetal bovine serum (heat inactivated)
  • DMSO (endotoxin free tissue culture grade)
  • Pipette and tips (wide-bore 1mL pipette tips, and standard tips)
  • Cryo-preservation container ("Mr Frosty" container or similar)
  • 15mL and 50mL conical bottom tubes
  • 37C water bath
  • Centrifuge (swing bucket)

Procedure

Blood collection

Prepare Wash Buffer: Phosphate Buffer Saline (PBS) without Ca2+ Mg2+ containing CTL-Wash™ Supplement.

  1. The BD Vacutainer®CPT™ Tubes should be stored at room temperature (18-25°C) upright.
  2. Draw blood into CPT tubes according to the product instruction.
  3. After blood collection, store CPT tube on racks upright at room temperature during transportation.
  4. Store tube upright at room temperature until centrifugation. Blood samples should be centrifuged within two hours of blood collection for best results.

PBMC Preparation

  1. If spiking in cancer cells, open blood tube cap carefully, spike in cancer cells, close cap completely.
  2. Remix the blood sample immediately prior to centrifugation by gently inverting the tube 8
    to 10 times.
  3. Centrifuge blood tube sample at room temperature (20-25°C) in a horizontal rotor (swing-out head) for a minimum of 20 minutes at 1500 to 1800 RCF (Relative Centrifugal Force).
  4. After centrifugation, mononuclear cells and platelets will be in a white layer just under the plasma layer.
  5. Re-suspend the cells into the plasma by inverting the unopened CPT Tube gently 5 to 10
    times.
  6. Open the CPT Tube and pipette the entire contents of the tube above the gel into a 15mL centrifuge tube.
  7. Rinse the CPT tube 2 times with 2mL PBS (with CTL wash) and add to the tube above. Add PBS (with CTL wash) to bring volume to 15mL. Cap tube. Mix cells by inverting tube 5 times.
  8. Centrifuge for 15 minutes at 300 RCF. Aspirate as much supernatant as possible without disturbing cell pellet.
  9. Gently tapping tube with index finger to loosen the cell pellet. Re-suspend the pellet using a p1000 pipette with a wide-bore 1mL pipette tip by slow and gentle pipetting until no visible cell aggregates can be observed (*cell pellet should be gently resuspended with this technique for all following steps in this protocol).
  10. Wash the cells once more by with 10mL of PBS. Cap tube. Mix cells by inverting the tube.
  11. Centrifuge for 10 minutes at 300 RCF. Aspirate as much supernatant as possible without disturbing cell pellet.

Freezing PBMC

Prepare Freezing Media: Make 20% FBS in RPMI and 20% DMSO in FBS. For rinsing make 10% DMSO in FBS. The following table iS an example of freezing media preparation per sample. Scale up as necessary.

*** Reports from other users indicated that Recovery™ Cell Culture Freezing Medium (Thermofisher Cat. 12648010) also works well. Fluxion has yet to validate this product.

Reagent 20% FBS 20% DMSO in FBS 10% DMSO in FBS
100% FBS 100µL 400µL 450 µL
100% DMSO      
RPMI 400µL    
Total 500µL 500µL 500µL
  1. Gently re-suspend the cell pellet in 20% FBS in RPMI and bring the volume to ~500µL
  2. VERY IMPORTANT: Add 500µL drop-wise of FBS containing 20% DMSO; gently swirl the
    tube after each drop.
  3. Gently transfer the entire content into a cryovial using the wide-bore pipette tip. Rinse
    the bottom of the 50mL conical tube with 200uL of FBS containing 10% DMSO. Add the
    rinse to the sample slowly.
  4. Close the vial and freeze according to the standard cell freezing procedure. We
    recommend slow freezing (using alcohol insulated container or equivalent) at -80°C
    overnight and transfer to liquid nitrogen after 24 hours.

Thawing PBMC

Prepare Thawing Buffer: Make RPM 1640 containing 1X CTL-Wash™. Keep warm at 37°C. Just prior to use, add Benzonase to a final concentration of 50Unit/mL.

  1. Remove the vial from liquid nitrogen and place in 37°C water bath until cells are 90% thawed. Remove from water bath.
  2. Using a wide-bore pipette tip, transfer the sample to a 50mL conical tube.
  3. Rinse the sample vial with 1mL of Thawing Buffer and add drop-wise the rinse to the conical tube, swirling the tube after each drop.
  4. Slowly add drop-wise 4mL of Thawing Buffer (with Benzonase) to the conical tube swirling the tube after each drop.
  5. Slowly add drop-wise 5mL of Thawing Buffer (with Benzonase) to the conical tube
  6. Slowly add 10mL of Thawing Buffer (with Benzonase) to the conical tube
  7. Gently swirl the tube. Incubate for 5 minutes at Room Temperature
  8. Centrifuge at 300 RCF at room temperature for 10 minutes
  9. Gently aspirate off the supernatant, being careful not to disturb the pellet
  10. Wash the sample once more with 10mL of Thawing Buffer. (If nuclease is not desired for down-stream analysis, use RPM containing CTL-Wash™ Supplement without Benzonase
  11. Centrifuge at 300 RCF at Room Temperature for 10 minutes
  12. Gently aspirate off the supernatant, being careful not to disturb the pellet
  13. Gently re-suspend the pellet in ~800µL IsoFlux Binding Buffer
  14. The sample is now ready for coupling for IsoFlux rare cell isolation