DNase I Treatment for Clumpy Cell Suspensions

Preparing a single cell suspension is critical for successful cell isolations. Performing cell separation on clumpy cell samples can result in lower recovery and may interfere with proper labeling of the target cells.

Samples may sometimes appear "clumpy" when they have been exposed to repeated freeze/thaw cycles or enzymatic tissue dissociation. These cell clumps occur because environmental stresses can accelerate the rate of cell death within the sample, resulting in the release of "sticky" DNA molecules from the dying cells that can clump neighboring cells together.

Adding the endonuclease deoxyribonuclease I (DNase I) into your sample can minimize the presence of free-floating DNA fragments and cell clumps. There are a number of valid protocols for DNase I treatment available to reduce cell clumping. We recommend adding DNase I Solution (Catalog # 07900) to the cell suspension at a final concentration of 0.1 mg/mL (or 200 Kunitz units/mL) and incubating for 15 minutes at room temperature prior to proceeding with downstream applications.

The following protocol for thawing frozen samples outlines how to prepare a single cell suspension and minimize cell clumping using DNase I:

  1. Thaw the vial of cells quickly by swirling it in a 37 °C water bath. Transfer thawed cells into a sterile 50 mL conical tube. Optional: Add 0.25 to 0.5 mL of a 1 mg/mL DNase I solution directly to the tube prior to transferring thawed cells.
  2. Rinse the vial with 1 mL of culture medium or buffer (i.e. PBS or HBSS) containing 10% FBS to recover any remaining cells, and transfer the medium to the new tube.
  3. Slowly add 10-15 mL of medium or buffer containing 10% FBS drop-wise, while gently swirling the tube.
  4. Top up the 50 mL tube with culture medium or buffer containing 10 % FBS. Gently invert to mix.
  5. Centrifuge the 50 mL tube at 300 x g for 10 minutes at room temperature to collect the cells (G to RPM conversion tool).
  6. Carefully remove and discard as much of the supernatant as possible, taking care to not disturb the pelleted cells. Gently tap the tube to resuspend the pellet.
  7. If cells appear clumpy, calculate the volume of DNase I that should be added to the sample to yield a final concentration of 0.1 mg/mL DNase I. Add DNase I drop-wise to the cell suspension while gently swirling the tube. Incubate for 15 minutes at room temperature.
  8. Add 25 mL of culture medium or buffer containing 2 % FBS, gently invert to mix and centrifuge as above. Discard as much of the supernatant as possible and gently resuspend the pellet.
  9. If cells still appear clumpy, pass the sample through a 30 - 70 µm mesh cell strainer into a fresh conical tube. Rinse the sample tube three times with culture medium or buffer containing 2 % FBS and pass through the strainer.
10. The cell suspension is now ready for cell counting and further downstream applications such as cell isolation.

NOTE: For downstream applications that are sensitive to DNAse (eg. hematopoietic colony assays), wash cells once in the appropriate assay buffer (without DNAse) before continuing.

For further assistance and information, please contact techsupport@stemcell.com.
For more protocols and tips for sample preparation prior to cell isolation, please visit the Human Immunology Portal.