Cord Blood CD34+ Cell Isolation

Technical tip from our dedicated team of Product and Scientific Support specialists

Every good experiment starts with the right cells. Whether you’re thawing frozen primary cells or selecting CD34+ cells from your own cord blood samples, knowing what you’re working with is key to your future success. In the case of isolating cord blood-derived CD34+ cells, great care must be taken to achieve high purity and recovery. Success with these samples can be limited by factors incurred through the cord blood collection process, and result in:

  • Low frequencies of CD34+ cells (typically 0.1 - 1% of nucleated cells) within each sample
  • Finite volumes
  • Variable cell quality and viability between samples

With the EasySep™ Human Cord Blood CD34+ Positive Selection Kit II (Catalog #17896) you can achieve CD34+ cell purities of up to 98% in less time than comparable column-based methods (see Comparison of Protocols below). This kit combines both RosetteSep™ lineage-depletion, prior to isolation of CD34+ cells with EasySep™.

Comparison of Protocols:

  1. Dilute sample and layer over density gradient medium.
  2. Centrifuge for 30 - 40 minutes.
  3. Remove the top layer and transfer to clean tube.
    Optional: Centrifuge for an additional 10 - 15 min to remove platelets.
  4. Filter and count cells.
  5. Centrifuge for 10 minutes.
  6. Incubate with blocking reagent and beads for 30 min.
  7. Dilute with buffer and centrifuge for 10 minutes.
  8. Aspirate supernatant and proceed to magnetic separation.
  9. Rinse column with buffer (~15 min*).
  10. Apply cell suspension to column & collect flow-through (~15 min*).
  11. Wash column with buffer & collect flow-through (~15 min*).
  12. Remove column and place on collection tube.
  13. Flush magnetically labeled cells into collection tube by pushing plunger into the column

Optional: Enrich sample further by running over a second column and repeating Steps 9 - 13.
  1. Incubate cell suspension with RosetteSep™ antibody cocktail for 20 minutes. This cocktail targets B cells, T cells, myeloid cells, red blood cells and platelets for depletion.
  2. Dilute sample and layer over density gradient medium.
  3. Centrifuge for 20 minutes at 1200 x g with the brake off (10 minutes with brake on using SepMate™).
  4. Wash pre-enriched cells with medium and centrifuge for 10 min.
  5. Resuspend pellet and incubate cells with EasySep™ Isolation Cocktail for 10 min.
  6. Add magnetic EasySep™ Dextran RapidSpheres™ and incubate for 1 minute.
  7. Place tube in magnet for 3 minutes.
  8. Pour off the supernatant and discard, positively selected CD34+ cells remain in the tube, repeat for a total of 4 x 3 min separations.
Total Time: ~2 Hours
*Column time is highly variable.
Total Time (with SepMate™): ~1 Hour
Total Time (without SepMate™): ~1.25 Hours

Benefits of this column-free, immunomagnetic method for the isolation of cord blood-derived CD34+ cells, as compared to column-based methods, include:

  • Less time, particularly for samples which contain dead cells and aggregates that may clog a column.
  • Less manipulation of the cells, fewer washes and less time that CD34+ cells are exposed to buffers and reactive surfaces.
  • Can provide significantly higher purity and equivalent cell recovery.

For optimal performance of the EasySep™ Human Cord Blood CD34+ Positive Selection Kit II (Catalog #17896) , please refer to the following tips:

Cord Blood Sample

  • Use freshly collected cord blood samples (ideally < 24 hours old).
  • Maintain cord blood samples at room temperature after collection and before processing (do not refrigerate).
  • Collect cord blood with Citrate Phosphate Dextrose (CPD) or Acid Citrate Dextrose (ACD) anticoagulant. Avoid the use of EDTA.

RosetteSep™ Pre-Enrichment

  • Layer the sample slowly and carefully on top of the density media in order to minimize of the mixing that can occur at the interphase.
  • After the RosetteSep™ spin, reduce the platelet contamination burden by aspirating some of the plasma layer off, being careful not to disturb the interphase containing the desired cells.

EasySep™ CD34+ Cell Isolation

  • During the magnetic separation steps, after decanting the supernatant, ensure the cells remaining in the tube are completely dislodged from the walls of the tube and suspended in buffer prior to the next separation. Tapping on the sides of the tube helps to loosen the cells which can be followed by thoroughly washing with EasySep™ buffer to obtain maximum cell yield.

To learn more about this and other kits for cord blood CD34+ cell isolation, view the our Technical Bulletin: Isolation of CD34+ Cells from Human Cord Blood . Not working with cord blood? We have more column-free methods for CD34+ cell isolation from bone marrow and whole blood. For further assistance with this product please contact

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