Hi,
what count cell is necessary for each slide?
what is the best program of cytospine for this sample?
what is the best fixation method for sputum cell ICC?
in acetone/methanol method, temperature of this solution must be 4 or -20 c?
Thanks
Usually between 20,000 to 32,000 cells should be placed per cytospin slide (0.5 cm approximate diameter). However, if the sputum contains a lot of squamous cells like in some cases happens when patients spit rather than expectorate, then you need to load the low value of only 20,000/well or even lower. Usually you spin 5 min at 1500 RPMs and that's enough. There's no single best fixation method, it depends on the antigen you're trying to detect. Because a sample of sputum will provide greater than 1 million cells, it's best to prepare several slides with different number of cells in duplicates (in the range pointed out before) and fix a few slides in fixatives like 10% formalin at room temperature for 15 min or Acetone/Methanol at -20 deg C for 15 min in the presence of proteinase inhibitors. - That way, you cover yourself and one of the conditions is bound to work.
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