I plan to work with signaling analysis in neutrophils. To do this, I need to isolate neutrophils, but instead of using a density gradient, I would like to perform a sorting separation by flow cytometry that will allow me to isolate the neutrophils and also obtain a specific number of cells in wells or tubes. However, given the sensitivity of neutrophils to activation, could the sorting process activate my cells?
I should mention that after the separation, I plan to incubate the cells with inhibitors and activators, as well as with specific antibodies for proteins that will allow me to assess signaling.
I would greatly appreciate your help.
Hello Marisol Alvarez-González
Yes, you are right. Flow cytometry sorting can activate neutrophils.
The mechanical stress while the cells pass through the flow, the type and concentration of antibodies used for labelling, bind to surface receptors can trigger activation signals as well as the conditions used during sorting like the buffer composition, flow rate and the pressure could influence neutrophil activation.
So, if you are going to perform a sorting separation by flow cytometry, you should optimize the experimental conditions. For instance, select those antibodies that are known to be non-activating, or you could use a lower concentration of antibody to minimize activation signals. Optimize the flow rate and pressure to minimize mechanical stress and activation. Also, I would suggest you include a control group in your experiment which is unstimulated so that you could assess the baseline activation state of neutrophils and compare with the sorted cells.
Best,
Hi Marisol,
I completely agree with all comments by Malcolm Nobre
He's given you very good advice.
Neutrophils are quite difficult to work with given their ability to get easily activated, and even induced to die.
When incubating with antibodies, also consider the temperature and the tubes. Neutrophils don't like low temperatures and they tend to adhere to plastic tubes, so use polypropylene tubes instead of polystyrene.
Taking advantage of multicolour flow cytometry, and if there are fluorochrome-conjugated antibodies available for the phosphorylated proteins you are interested in, you could do your experiments with total blood, without sorting.
If you really need to sort your cells, to minimise the flow stream and nozzle effects, consider using a low-pressure sorter. One good system is the WOLF G2:
https://nanocellect.com/wolf-g2-cell-sorter/
All the best!
Malcolm Nobre and Fabian Flores-Borja
I truly appreciate your comments and recommendations. As you suggested, I think I’ll standardize the technique using cell sorting to evaluate whether there’s increased activation due to external factors. If that’s the case, I’ll definitely consider minimizing cell manipulation. Thank you all!
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