Research Flow & Imaging
Cytometry Resource

Cell Sorting FAQs



What should I bring my samples in for sorting?
Samples should be brought in 12x75mm Falcon tubes (catalog# 352008). NOTE: same size tubes by VWR or other manufacturers may not fit the sample port of cytometer.For collection tubes you can also use 12x75 mm Falcon tubes. For abundant populations we recommend 15 ml Falcon tubes. We can also sort into a range of microtiter tubes, slides and Petri dishes. Sorting of small amount of cells can be done in siliconized Eppendorf tubes (1.5 ml).

How many cells do I need bring for cell sorting?
The answer is related to following information: 1/ what is the approximate percentage of the smallest population you wish to sort?; 2/ how fragile and large are your cells?; 3/ how many cells do you want to get back? 4/ do you want enrichment or purification of population of interest?

What controls do I need?
Unstained cells, along with positive and negative controls, are needed.   Positive controls are used for compensation of fluorochrome emission overlap. Compensation beads from BD Biosciences labeled by your antibody conjugate may be used instead cells. Negative controls may consist of unstained samples, isotypes, and fluorescence minus one (FMO) flourochrome staining. If you choose to bring isotype controls, then isotypes should be of the same subclass, species and fluorochrome, as the antibodies used in the experiment.  The isotypes should also be used at the same concentration and F:P ratio as the specific antibody and preferentially purchased from the same manufacturer. For sorting of FP (fluorescent protein)-transfected cell lines we prefer to have untransfected cell line as control.

Do I have to fix my cells? If so, what do I fix them in?
 Any samples which contain potentially biohazardous material should be fixed unless you request and discuss with us biohazardous sort. Cells should be fixed in a final concentration of 1-2% paraformaldehyde in PBS. You may also consider using Cyto-Chex for fixation.

Do I need to filter my cells?
Yes, It is REQUIRED that your cells be filtered, preferably right before running on the sorting. We are asking you to use 40 u filter for FACSAria sorts.

What do I resuspend my cells in for cell sorting?
After the final centrifugation (wash), cells should be re-suspended at concentrations of 10-20 million for primary cells or 5-10 million for cell lines per ml in 12x75 mm Falcon tubes in PBS with 0.5% FCS

How long will cell sorting take?
It depends from the nature of your cells (fragile and large cells need to be sorted at low pressures and speeds and may require larger nozzle) and the concentration of your sample (diluted samples will take longer run).

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The Program in Cellular and Molecular Medicine (PCMM) is an affiliate of Children's Hospital Boston and Harvard Medical School.