Preparing a single-cell suspension is critical for successful cell isolations, but if your cell samples are clumpy, it can result in lower recovery and may interfere with 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. This protocol describes how to reduce cell clumping in single-cell suspensions by treating your sample with DNase I.

Materials

• DNase I Solution (1 mg/mL, Catalog #07900)
• Culture medium or buffer free of EDTA (e.g. HBSS, Modified (Without Ca++ and Mg++), Catalog #37250, or phosphate-buffered saline [PBS])
• Fetal bovine serum (FBS)
• Two 50 mL conical tubes (e.g. Falcon® Conical Tubes, 50 mL, Catalog #38010)
• Cell strainer (e.g. 70 µm Reversible Strainer, Large, Catalog #27260)
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• PBS containing 2% FBS (e.g. Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum, Catalog #07905)
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)
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Protocol

1. Quickly thaw cell vials in a 37°C water bath or the ThawSTAR® CFT2 Automated Thawing System for ensured sample sterility and consistent thawing performance. Transfer thawed cells to a sterile 50 mL conical tube.
   Optional: Using a pipettor, add 0.25 to 0.5 mL of DNase I solution directly to the tube prior to transferring thawed cells.
2. Slowly add 10 - 15 mL of medium or buffer containing 10% FBS dropwise, while gently swirling the tube.
3. Rinse the vial with 1 mL of culture medium or buffer (e.g. HBSS or PBS) containing 10% FBS to recover any remaining cells, and transfer the medium to the new 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 (15 - 25°C) to collect the cells.
6. Carefully remove and discard as much of the supernatant as possible, taking care to not disturb the cell pellet. Gently tap the tube to resuspend the pellet.
7. If cells appear clumpy, calculate the volume of DNase I Solution that should be added to the sample to yield a final concentration of 100 μg/mL DNase I. Add DNase I Solution dropwise to the cell suspension while gently swirling the tube. Incubate at room temperature for 15 minutes.
8. To wash the cells, add 25 mL of culture medium or buffer containing 2% FBS. Gently invert to mix, then centrifuge at 300 x g for 10 minutes at room temperature. Discard as much of the supernatant as possible, then gently resuspend the pellet.
9. If cells still appear clumpy, pass the sample through a 37 - 70 µm cell strainer into a fresh conical tube. Rinse the sample tube three times with culture medium or buffer containing 2% FBS, then pass through the strainer.
10. The single-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 (e.g. hematopoietic colony assays), wash cells once in the appropriate assay buffer (without DNase) before continuing.