I am performing a phagocytosis assay on mouse peritoneal cavity cells and detecting them using flow cytometry. Since the cells remain on the culture plate for 5 hours during the assay, they become strongly adherent. Scraping them in 2 mM EDTA for flow cytometry is causing significant damage—I see most of the cells appearing as debris in the FSC/SSC plot. I’m using non–tissue culture–treated plates.
What else can I do to harvest the cells without damaging them? Do you think performing flow staining directly on the plate and then scraping the cells after fixation would help reduce cell damage?
Dear Dr. Vahinipriya Manoharan
Yes. For adherent cells that are difficult to detach like the macrophages, you should try staining on the plate as it can result in better cell recovery and viability compared to methods that cause cell damage during detachment. This method can help preserve cell surface markers and potentially reduce cell damage compared to enzymatic or any other harsh detachment methods.
I have provided a brief protocol below.
1. Culture macrophages in plate or dish, ensuring that they are properly adhered.
2. Aspirate culture media.
3. Wash the cells with PBS.
4. Add the desired antibodies diluted in a staining buffer (e.g., PBS with 0.1% FBS or BSA) directly to the cells in the plate/dish.
5. Incubate for the recommended time and temperature (on ice and protected from light).
6. Wash the cells with staining buffer to remove unbound antibodies.
7. If you need to perform intracellular staining, include the fixation and permeabilization steps.
8. After fixation, gently detach the cells from the plate/dish using either cold PBS with 5mM EDTA or accutase. Alternatively, scraping can be used after fixation, but be a little careful not to damage the cells.
9. Transfer the detached cells into flow cytometry tubes and proceed with flow cytometry analysis.
Regards,
Malcolm Nobre
Hi Malcolm,
Thanks for your message. My concern is regarding step 8. Do you think fixing the cells before scraping would cause less damage to the cells? I’ve attached the flow graph of cells that were scraped and then fixed—you can see the debris. Do you think fixing the cells first and then scraping would help reduce that?
Hello Vahinipriya,
I have not performed such an experiment, but it should help because fixation, particularly with formaldehyde, will stabilize cell membranes and prevent cell damage during the scraping process, especially for adherent cells. So, a more stable cell structure may lead to fewer cells being lost during the scraping step, which is crucial for obtaining accurate data in flow cytometry.
You should try it once, and I would be happy to know the results in case you decide to perform such an experiment.
Good Luck!
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