This is the most commonly asked question. To answer it we need to know the following information: What is the approximate percentage of the population(s) you wish to sort? How many total cells do you want back? Do you want stringent purification or enrichment? How fragile or large are your cells? All of these [...]
The answer to this question depends primarily on your goal (enrichment vs stringent purification), the nature of your cells (fragile cells or large cells need to be sorted at lower pressures and speeds with a larger nozzle), and the concentration of your sample (more dilute samples will take longer to sort). For example, on [...]
It is REQUIRED that your cells be filtered through at 70um nylon mesh, preferably right before running on the sorter. We can provide the mesh if necessary.
Proper sample preparation is absolutely essential for obtaining good purity and yields. After the final wash, cells should be re-suspended at concentrations of 20-30 million (for primary cells) or 5-10 million (for cell lines) per mL in FACS Tubes (see below). The media can be any buffered isotonic salt solution (such as PBS) but [...]
You will need to bring an unstained control, single colour compensation controls and you MAY need to bring FMO (Fluorescence Minus One) gating controls. You may want to consider an FMO control if you are using more than 5 colours simultaneously and if your populations of interest cannot be discretely separated from background/negatives. Please [...]
For abundant populations we recommend 50 mL (MoFlo) or 15 mL (FACS Aria) Falcon Tubes, or 5 mL Falcon tubes. We can also sort into a range of microtiter plates, including from 96-well to 8-well, microscope slides or Petri dishes. Please talk to us about your requirements and we will advise you on the [...]
We recommend 100% serum or HEPES buffered HBSS. The sorter uses PBS as its sheath fluid. Mixing large quantities of PBS with buffer containing calcium chloride may produce a precipitate if calcium phosphate on the cell membranes. This can adversely affect viability. For this reason, it is preferable to sort large numbers of cells [...]