I am currently running Cell Death ELISAs, using the Roche Cell Death Detection ELISA kit. My samples are from the supernatant of bacterially infected primary canine cells. I am seeing no color change in my samples OR my postive control. The Positive control should read at at least 600mU (absorbance) within 15 minutes of substrate reaction.
Could anyone provide some insight or guidance?
Ensure that all reagents, including the substrate, are prepared correctly and have not expired. Incorrect preparation or expired reagents can lead to no color development. Verify that your positive control is prepared correctly and is active. If the positive control doesn't show the expected absorbance, there might be an issue with the kit or the reagents.
Double-check that you are following the recommended incubation times and conditions precisely. Inadequate incubation can lead to insufficient color development. Make sure the substrate is added last and that it is protected from light, as it can be light-sensitive.
Confirm that the microplate reader is properly calibrated and functioning correctly. An issue with the reader can result in inaccurate absorbance readings.
Ensure that your samples are prepared and stored correctly. Degradation of the samples can affect the results. Verify that the washing steps are thorough and consistent. Inadequate washing can result in a high background or no signal. If you have checked all the above points and still face issues, consider contacting Roche technical support for further assistance. They might be able to provide additional insights or replace the kit if it's defective.