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Low vehicle controls in Neutral Red assay

cytotoxicity Neutral Red

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#1 jn315

jn315

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Neutral

Posted 25 July 2012 - 12:41 PM

I have been having issues with the vehicle controls in my NRU assays. They must have an OD540 of >0.3. Lately, the readings are between 0.20 and 0.27. I have been using the Fisher NR (cat # N129-25). I ordered some from Sigma (cat # N4638) and tried that. With the Sigma NR, my readings were even lower. The cells look fine, before adding NR, they are ~90% confluent, morphologically normal and there are still some mitotic cells present.

When I dilute the NR for use, it is in basal DMEM (no serum, antibiotics, L-glutamine) and incubated overnight at 37C. Just before use, I filter it using a Millipore 0.2 micron filter. Also, my media does not contain phenol red.

Does anyone have any helpful hints? My Fisher NR is about 3 years old and has been stored appropriately.

Thanks!





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