I treated the cancer cell to induce calreticulin(CRT) exposure on the cell surface. Before I could observe 20% of ecto-CRT(+), then it dropped to 5%, then it dropped to nothing recently. I checked the cell contamination, used new the batch of cells, the new batch of drug, and also the new vials of antibody. But I couldn’t think any other reasons why I couldn’t get duplicated result. Also to clarify, the flow cytometer was validated and passed the performance test every time.
Here is my protocol. 1 million cells were cultured in 6 cm plate and performed the drug treatment. The cells were harvested by Accutase buffer. Wash twice with ice-cold PBS, the cells were blocking with 1% BSA-PBS. Then incubated with monoclonal mouse anti-CRT antibody (1ug/100ul) on ice for 1hr. Wash once. Incubated with goat anti-mouse IgG (Alexa fluor 488) on ice in the dark for 1hr. Wash once. Diluted the sample in PBS and tested by flow cytometer. I'm using the scatter plot to gate the live cells since the drug mitoxantrone has fluorescence ~600nM which is overlapped the PI cell viability dye.
Hi Yajing Sun
It seems to me you have a plotted a too permissive gate so you get negative events included as positive.
you will get better results if you use a more strict gating strategy.
I've attached your plots with my gating suggestion marked in red.
Good Luck
Dear Yajing Sun,
As Avishay Edri suggested, there seems to be a problem on the negative controls. According to your data, MTX substantially affects the cell morphology and this could cause different background signal levels in the Alexa 488 channel. If this is the case, then you should not use untreated cells as a negative control for background fluorescence levels.
A quick hack for the situation is to compare (1) MTX-treated unstained cells and (2) MTX-treated ecto-CRT-stained cells for the Alexa 488 signal levels. If you do not find substantial signal up regulation in (2), then perhaps you have failed to induce CRT.
If you do find a significant signal level difference between (1) and (2), then I would suggest using the same MTX-treated cells stained with Alexa 488-conjugated isotype control antibody as a negative control.
Another thought is that you should always include viability dye. There are many available, which can overcome your overlap with PI. Depending on the lasers available on your machine, you could use DAPI or 7-AAD. These work the same as PI. Another option would be the fixable live/dead amine-reactive dyes. These are available in a variety of colors from eBioscience, BioLegend and Invitrogen. Good luck.
I also agree with the updated gating above.
Dear Yajing Sun,
I think you might have a problem with your negative control, maybe you are not using the right one. Besides that, I suggest you to change your gating strategy because it seems not strict and it can inlfuence your results including negative events in positive events. Therefore, the results are biased.
Hello Inês Lua Freitas Allison C Sharrow Tetsuo Tsukamoto Avishay Edri
Thank you so much!!
I took your suggestion and used PI as the cell viability dye. With the low concentration of the MXT, the fluorescent overlap was minor. I tested with the unstained control and 1uM MXT samples. I don't know whether I could switch to another negative control because the purpose was to compare expression of ecto-CRT on treated and untreated cell.
The cells were gated in this way: all -->singlet -->PI(-) --> ecto-CRT(+). Even the population of ecto-CRT+ was small, and there was a difference in MFI of CRT-Alexa 488 between control and treatment. It’s consistent with the published data. I hope my analysis was proper. Please correct me if I make any mistake.
I would not recommend to "switch" to another negative control. Rather, I recommended to add another type of negative control.
When you think of negative controls, it is important to identify what each one of them is negative for.
Your current negative control is "negative for MTX". This did not work for separating real CRT expression and background signal up regulation caused by the MTX treatment. To troubleshoot the problem, I recommended a control that is "negative for antibody specificity".
I would recommend placing isotype control antibody-stained samples for both your MTX treated sample and MTX-negative control sample. This even allows you to measure the CRT expression level in the MTX-negative control sample.
By the way, titrating MTX for your data would be another issue and come after you have settled the control problems.
Tetsuo Tsukamoto
Thank you for your prompt response! Yes, I should have an isotype control. before I only used the untreated control and 1uM MXT with secondary antibody only.
So far it is hard to said what CRT positive control looks like. Most of papers only reported the histogram, and there was no fully separating between untreated and treated samples. Maybe the expression of CRT is limited in the real world. I'm thinking to change the cell line or treatment.
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