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To ensure the best possible outcome for your cell sorting experiment, please include the following (where applicable) in your sample preparation protocols

RED BLOOD CELL LYSIS –Red blood cells can cause the most dramatic shift in efficiency and purity of sorting due to the high prevalence and resulting percentage shift of your cells of interest. When applicable please perform this step during your initial processing.

PRE-SORT PURIFICATION KIT– If your cell population of interest is a sub-optimal percentage (< 1% of total events) within the sample you can increase the efficiency and purity of the sort by potentially using a MACS cell separation technique. This could be useful for samples that have a high number of dead cells and debris, or for isolating a specific upstream lineage marker. (For example: perform a positive bead purification on PBMCs for CD4+ T cells to sort highly pure subsets)

MEDIA/BUFFER – Cells can be prepared in any colorless media or buffer that does not contain protein which can react with staining dyes or LiveDead dyes causing high levels of background fluorescence. Adding EDTA to your sample media can help prevent clumping. When culturing cells, although we sort in sterile conditions, it is still strongly recommended that you treat your media with PennStrep 3 days before, the day of, and 3 days after sorting to prevent any possible contamination. Fc block is also highly recommended in your staining buffer in order to prevent non-specific binding of your antibodies resulting in false positives.

CONTROLS – Controls are required to properly set up your sort. Please bring compensation controls (a true negative/unstained control and single-color controls for each fluorochrome in your experiment) in order to calculate proper compensation of spectral overlap. FMOs (Fluorescence Minus One) can be very helpful to set up gating and is especially the case for very rare populations or populations that tend to have a smear like stain. Isotype controls can be used to determine non-specific binding/background of your antibody stain.

LIVE/DEAD DYES – Using a Live/Dead dye on your samples can help to make sure that sorted cells are live and intact at the time of sorting, which can be especially important in certain post-sort applications. There are 2 types of dyes to choose from: Nucleic Acid (DNA) binding (PI, DAPI, 7AAD, etc) vs. Surface Protein (amine reactive) binding (LD, Zombie, etc). They both work by binding dead cells and gating negatively on the live population.

NOTE: Large and/or granular cells types tend to be more difficult to discern from dead cell expression levels due to a high level of background fluorescence of those cells. It’s important to test concentrations of your dyes to achieve the best separation possible with respect to the cell types you are using. When using Surface Protein Live/Dead dyes, this issue of overlap can increasingly become a concern especially when comparing a sample comprised of a mix of cell types, sizes, and granularity. These amine reactive dyes produce a “relative expression” as they are intended to stain both live and dead cells but to varying degrees based on the available proteins with amine sites on them. It’s understood that when comparing the same cell types, you will see a distinct positive and negative fraction due to the relatively higher level of amine sites in a permeabilized dead cell than a live cell with only amine sites found on the cell surface. However, when comparing the relative available amine sites between a mix of cells with different sizes, shapes, and granularity, the resulting stain can turn in to one indistinct smear. This can lead to gating in such a way that either misses a portion of a large-sized live cell population (or high background fluorescence population) to avoid dead cells or for sake of including all cell types/sizes/granularity, does not avoid including some dead cells in the gate. Please select your LiveDead dyes based on your specific experiment goals and sample types while understanding these limitations.

CONCENTRATION – Please prepare your cells at 15-20 million/mL, but in no less than 200uL volume.

FILTRATION – Samples MUST BE FILTERED no more than 30 minutes before the start of your reservation. Please use 35 um filter cap FACS tubes if available for 70um & 85um nozzle configuration and 70um filter mesh for 100um nozzle configuration.

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