Dear everyone,
I want to label THP-1 cells for IF microscopy but cannot find any detailed protocol on how to label suspension cells without cytospin - is there at all a way of labelling suspension cells without using a cytospin?
I tried to fix, permeabilize, block, and label the cells in suspension in eppendorf tubes with centrifugation in between all steps. But after permeabilization with 0.1 % Triton X-100, I don't get the cells to sediment anymore (I even tried to use 16000 xg in a tabletop centrifuge).
So, any suggestions how I can stain the cells? I would be very greatful for a detailed explanation on how to do it. Thanks a lot in advance!
Lena
Hello Lena Gauthier
You can refer to the protocol in the article attached.
Article Protocol for adhesion and immunostaining of lymphocytes and ...
Immunostaining of non-adherent cells is commonly performed after adhesion of cells onto microscope slides either using cytocentrifugation or with the help of charged coating substrates. These techniques, however, require either specialized equipment or significant preparation time. Here, we describe a method for immunofluorescent staining of lymphocytes within multi-well culture plates, where cells suspended in phosphate buffered saline (PBS) are adhered to either the plastic well bottom or glass coverslips by gravity sedimentation. This technique requires only common laboratory materials, no coating steps, and allows for densely adherent cell coverage with 1 × 106 cells. Our data show that suspension of cells in PBS, but not serum-containing growth medium, allows for adhesion to plastic or glass after 30 min of gravity sedimentation. We show that this method is applicable for immunofluorescent staining of both primary human lymphocytes and immortalized lymphoma cells, and that it preserves cell morphology.
Please refer to the protocol in the supplementary data.
https://www.future-science.com/doi/suppl/10.2144/000114610/suppl_file/Tsang_Supplementary_255323a.pdf
Hope this is helpful.
Good Luck.
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