Anti-Clumping Agent, 20 mL - FAQs

Use anti-clumping agent at a dilution of 1:250 to 1:1000 in culture medium. Test for impact on the cell culture and reduce concentration as the cell clumping is reduced.

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When cells meant to be grown in suspension are grown in static culture, they may form clumps. These clumps will severely limit transfection efficiency and protein expression. It is suggested that FreeStyle 293 cells in FreeStyle media and CHO-S cells in CD-CHO or CHO-SFM are grown in agitated suspension to reduce the appearance of clumps. However, if clumps do form, you can try the following protocol to select for cells that don't form clumps:

- Transfer cells into an appropriate size centrifuge tube that will hold the entire cell suspension.
- Allow cells to sit undisturbed for about 5 minutes. The time can vary depending on the specific cell line. Larger cell clumps will settle to the bottom of the tube.
- Collect cells from the upper portion of the tube to passage into a new flask.

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There are a number of factors that can contribute to this:<

1) Presence of calcium and magnesium ions. Wash cells in a balanced salt solution without calcium and magnesium. Gently pipette cells to obtain a single cell suspension.

2) Mycoplasma contamination. Segregate culture and test for mycoplasma infection. Clean hood and incubator. If culture is contaminated, discard.

3) Cell lysis and release of DNA resulting from overdigestion with proteolytic enzymes. Treat cells with DNase I.

4) Certain suspension cell type try to aggregate when they are not under optimal agitation speed and optimization is required.

5) Certain suspension cell type try to aggregate in bioreactors and it is recommended to use Anti-Clumping Agent (Cat. No. 0010057AE).

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Please use this selection chart that compares our cell dissociation reagents (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin.html).

Find additional tips, troubleshooting help, and resources within ourMammalian Cell Culture Basics Support Center.