Cell Preparation
Cells pass through a 70µm nozzle tip on the instrument, so a single cell suspension is essential. Large cell clumps will settle out of a cell preparation in 10 minutes at 1 g. Please filter cells through a 50µm nylon mesh (PGC Scientific Corp.) after all staining procedures to remove clumps and debris. Clumps can clog the instrument fluidics and in some cases divert the waste stream into the collection tube, thus contaminating the sorted cells and ruining the entire experiment up to that point.
Media
You will need two types of media:
- Media to sort or analyze from
- For experiments not requiring culture or functional analyses on sorted cells: Sorter Media is Calcium and Magnesium-free Hanks Balanced Salt Solution containing 0.1% NaN3, 0.2% BSA, 0.744 g/L EDTA and 5 x10-4 g/L Phenol Red. Sorter medium is available commercially from Quality Biological Inc.
[Note: this low level of Phenol Red is to provide a visual indication of a pH change of the stock medium, while staining or washing cell suspensions. Do NOT use high phenol red media such as RPMI or DMEM to resuspend cells for flow cytometric analysis. Phenol red at concentrations used in these media will produce higher background autofluorescence, particularly in high wavelength FL2 or FL3 PMTs.]
- For experiments requiring culture of or functional analyses on sorted cells: PBS, HBSS or RPMI with 5 – 105% FCS and Pen/Strep antibiotics but without phenol red.
- Media to sort into—Same as above. For sterile sorting this should be culture media, e.g., RPMI, with antibiotics but without phenol red.
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