The disappearance of the CD8a signal in your FACS experiments can be quite puzzling, especially when you haven't made any apparent changes to your protocol or reagents. However, troubleshooting such issues can be complex and require a systematic approach. Here are a few suggestions to help you identify the problem:

1. Antibody issues: Even though you mentioned using reagents from the same companies, it's possible that the antibody specific to CD8a may have changed or become less effective. Ensure that you are using the correct antibody clone and check its expiration date. Consider trying a different CD8a antibody from another supplier to see if it resolves the issue.

2. Instrument issues: While you have been using the same cytometer for years, technical problems can still arise. Make sure all lasers are functioning properly, and check the instrument's settings, such as voltage, compensation, and filters, to ensure they are correctly configured for CD8a detection. Clean the flow cell and other components of the cytometer, as debris or contaminants can affect signal detection.

3. Cell preparation issues: Verify that the tumor dissociation and staining processes are being performed consistently and accurately. Changes in dissociation enzymes, dissociation time, or staining conditions can influence the CD8a signal. Ensure that the cells are properly fixed, permeabilized (if required), and stained with appropriate controls.

4. Cell quality issues: In some cases, the loss of CD8a signal may be due to changes in the cell population you are analyzing. It is possible that the CD8a-positive cells are not present or are present at lower frequencies in the tumor samples. Try analyzing samples from different time points or conditions to determine if the loss of CD8a signal is consistent.

5. Other factors: Although you mentioned that nothing changed, it's worth considering other factors that might affect the CD8a signal, such as changes in the mouse strain, genetic modifications, or environmental factors. Additionally, double-check if any changes were made to the software or analysis settings of the flow cytometer that could impact the CD8a detection.

All the best

Hi! Thanks

1. Same antibody as in the past, same clone and everything. On other organs we see a nice binding- spleen, blood, LN...

2. We changed 2 filters 5 months ago, but it worked for a month after.

The maintenance guy was here and checked the machine and said that it's all good. If that was the issue, maybe we wouldn't get signal in other organs..

3. I haven't changed anything in the protocol and it worked for me for 2 years, I tried fixed samples, non-fixed samples, it's really not consistent.

4. Since I use those same tumors for 2 years already, I usually get around 15%-20% cd8 t cells at this time point, now I get zero..

5. I haven't changed the strain, I'll check the other points though

Thank you for your answer, any other ideas?

Based on the additional information you provided, here are a few more ideas to consider:

By systematically investigating these potential factors, you may be able to identify the cause of the CD8a signal loss in your FACS experiments.

All the best

Thank you so much :)