General considerations.

General Considerations:  The more cells you have, the better! For adherent cells, this protocol is not worth doing unless you have at least one confluent T175 (175 cm) of cells. There is variation in cell size and density, so for some cell lines even a T-175 may not give an adequate cell yield; thus, if it is little added trouble or expense it is best to shoot for 2 T175 flasks worth of cells.  Some cell lines will naturally form thick and stable pellets, others won’t. There’s almost no way to tell how well a cell line will pelletor how big the pellet will beuntil you try it.  The final step involves slicing the microfuge tube with a razor blade or scalpel. Exercise caution here!  Remember that when your fixed pellet goes in for processing with ethanol and xylene, it will shrink significantly (by about 30-50%). This means that if your initial pellet is 5 millimeters thick, after processing it will only be about 2.5-3 mm. Again, more cells/bigger pellets to start with are preferred.  We typically fix in 10% neutral buffered formalin at room temperature for 24 or 48 hours. Not sure that this is optimal for all purposes.  The size of the microfuge tube can be changed, depending on the expected size of the cell pellet. We generally use 0.5 ml PCR-type tubes. <0.5 ml size tubes can be used if the cell pellet is small.  A useful option for too few cells is to admix your cells of interest with a bolus of carrier cells (e.g. an easily grown cancer cell line). We have had very good success with this modification. Best strategy is to select a carrier that is significantly different than your cells of interest, especially for the particular marker(s) you will be trying to stain for, so that the two cell types will be easily distinguished from one another following staining.