How to Reduce Cell Clumping in Single-Cell Suspensions Using DNase I
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)
Protocol
-
Thaw the vial of cells quickly by swirling in a 37°C water bath. 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. - Slowly add 10 - 15 mL of medium or buffer containing 10% FBS dropwise, while gently swirling the tube.
- 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.
- Top up the 50 mL tube with culture medium or buffer containing 10% FBS. Gently invert to mix.
- Centrifuge the 50 mL tube at 300 x g for 10 minutes at room temperature (15 - 25°C) to collect the cells.
- 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.
- 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.
- 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.
- 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.
- 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.
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