May some of you performed staining for immunofluorescence of PBMCs in 96-well plate? As the cells are not going to be cultured, only stained in these plates for confocal microscopy, we are planning to centrifuge (600 g, RT, 6 min) cells in 96 well plate to attach them to the surface of the plate, however I am not sure if this is enough to attach them. I know there are coating plates, but for decreasing costs we are searching alternatives.
Dear friend Carlos Alejandro Bello Rodríguez
Attaching PBMCs to a 96-well plate can be challenging due to their low adherence properties. However, there are a few methods that can be used to improve cell attachment.
One method is to use poly-L-lysine (PLL) or fibronectin coating of the wells. PLL and fibronectin are positively charged and can help PBMCs attach to the negatively charged surface of the wells. Another method is to use a centrifugation step, as you mentioned in your question, to force the cells to adhere to the plate.
In addition, some researchers have reported success with treating the wells with ethanol or methanol prior to seeding the cells, as this can remove any potential contaminants on the surface and increase attachment.
Here are some references that may be useful:
1. Zhang, S., Li, X., Li, H., Li, Y., Li, L., Li, Q., ... & Li, D. (2018). Poly-L-lysine improves cell attachment and proliferation on electrospun polycaprolactone fibers. Journal of Biomaterials Applications, 33(2), 266-276.
2. Hill, M., & Parry, H. (2014). Adherence of lymphoid cells in microculture. Journal of Immunological Methods, 406, 7-13.
3. Lee, S. J., Cho, K. J., Lee, J. Y., Cho, J. Y., Lee, J. H., Kim, J. H., ... & Cho, Y. S. (2011). Development of an immunofluorescence assay for the detection of Plasmodium vivax using a monoclonal antibody against PvMSP-1. Korean Journal of Parasitology, 49(3), 251-257.
Dear Kaushik Shandilya, thanks for your reply and suggestions. I was trying to find the references that you mentioned, however I could not find them. Do you have the doi which could help. Thanks
Dear friend Carlos Alejandro Bello Rodríguez
I think you need to connect with your library or google scholar or approaching the authors will certainly help you
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