FAQs for Cell Sorting

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FAQs for Cell Sorting

What types of cells can I sort?

Any type of cells, which can be easily kept in single cell suspension, can theoretically be sorted. Cells that easily clump, agglutinate or settle out of suspension can be problematic when sorting, as this will cause clogging of the nozzle and disturbance of proper droplet formation. Primary Human samples or samples infected with BSL2 pathogens require special handling and engineering precautions. Please contact Joanne Lannigan (924-0274) for special instructions prior to scheduling these samples.

 

What are the advantages of sorting by Flow Cytometry compared to other isolation techniques (i.e. magnetic bead separation, panning, etc...)?

a) Higher purity
b) Can separate populations of cells based on their level of fluorescence intensity
c) Better separation of populations using multiple antibodies
d) Higher recovery of cells of interest
e) Can eliminate dead cells
f) can sort based on internal staining (DNA, cytokine expression, GFP, etc.)
g) can sort up to four populations simultaneously

What effect will the sorting process have on my cells' viability?

The effects of sorting on cells is dependent on several factors:
a) cell type - some cell types are more "fragile/labile" than others. Any type of cell that has structure on the outside of the cell membrane is more likely to be damaged during the sorting process.
b) condition of the cells prior to being sorted - cells that have been activated or treated with drugs may be more susceptible to the effects of sorting. Apoptotic cells are likely to be selected against under higher pressures
c) pressure/jet velocity at which cells are sorted - the faster you want to sort, the higher the pressure. Some cells (i.e. DCs) do not do well under high pressure conditions. Faster is not necessarily better.
d) the buffer the sample is to be sorted into - most cells will not tolerate significant mixing of buffers. Carbonate buffers when mixed with phosphate buffers (sorting fluid) can cause precipitates to form on cell membranes.

How many cells do I need to bring?

This will depend on a) how many cells you need to recover; b) the frequency of the target population of interest and c) % yield (generally 75-90%). For example, if the target cells you are interested in sorting are 10% of your unsorted cells and you need to recover 1 x 106 target cells, you would need 2.0 x 107 as a starting cell number:

[2.0 x 107 x 0.10 x 0.50 = 1 x 106 ]


It is always best to calculate the number of input cells based on the worst-case scenario of a 50% yield rate so you will be sure to have an ample number of cells to start.

 

In order to have a successful sort and to properly configure the instrument, the following information is required at the time of reservation (under details section):

a) What type of cells (species, tissue, primary or cell line); this is important for establishing instrument settings and pressures
b) What size are the cells; this is very important in selecting the nozzle size to be used
c) What has been done to the cells (stimulated, drug treated, transfected); this is important in choosing nozzle size and jet velocity
d) Which fluorochromes have been used to label the cells; this is important in selecting lasers and optical filters and in setting compensation for spectral overlap. It is very important to discuss these issues with the operator prior to setting up your experiment to insure the instrument has the ability to excite, detect and compensate your fluorochrome combination.
e) What will be done with the sorted sample (short-term culture, long-term culture, PCR, functional assays, etc); this will help determine the sterility conditions as well as jet velocity.
f) Vessel type you wish to sort into (i.e. 12x75mm tube, 15ml Conical Tube, 96 well plate, 24 well plate, 6 well plate, microscope slide)
g) Number of populations to be sorted (1-4 populations). Note: Only one population can be sorted for plate and slide sorting.

What is yield and why is it only 75-90%?

Yield is defined as "the {[number of cells in the collection tube] X [the % purity]} /{ [the number of cells you started with] X [% of the target population]}. There are a number of factors that affect the actual yield of the sorted sample:

a) electronic aborts - cells which arrive in the laser beam too close to one another; with each electronic abort, more than one cell is thrown away and not processed
b) sort conflicts - a target and non-target cell occurring within the same or overlapping sort envelopes; both target and non-target cells are not sorted.
c) Loss of target cells due to cell death (pre and post sort) or adherence to tube walls

Increasing sample rate (#cells/second analyzed) and the presence of cell aggregates can increase these losses and ultimately reduce your yield.

How can I keep my cells from clumping?

Keeping cells in single-cell suspension during the entire sort is critical to the success of a sort. Cell preparations with a large number of cellular aggregates can cause the nozzle to clog and necessitate the re-establishment of instrument setting. Nozzle clogs can also increase the possibility of sort contaminations. Cell clumping can be a problem with adherent cells, activated cells, or samples with a high percentage of dead cells. Raising the concentration of EDTA to 5mM in the sample buffer may help with reducing cation-dependent cell-to-cell adhesion. For adherent cells, there are commercially prepared cell detachment products, such as Accutase, or the use of cation-free FBS buffer as a Trypsin inhibitor, can help reduce clumping. If there are a large number of dead or damaged cells in the sample, soluble DNA can cause the cells to become "sticky" and start to clump. The addition of DNAase II (10U/ml) to the buffer solution can help eliminate the clumping due to free DNA. If, in spite of these efforts, clumping still occurs the sample should be passed through a nylon mesh to remove as many clumps as possible prior to the sort (Sample Preparation).

How long does an average sort take?

This is dependent on how many cells you need to recover and the % of target cells in the original sample. If you are sorting for a rare population of cells, it will generally take much longer than if you are sorting a population that is 30-50% of your original sample. 2.0 x 107 cells can take anywhere from 1.0-2.0 hours, depending on the pressure, quality of sample, and size of the cells (larger cells require a larger nozzle which necessitates lower pressures). Set up time for a sort takes about 60-90 minutes, 10-15 minutes to establish regions and sort gates, and 10-15 minutes for post sort analysis.

How many populations can I sort simultaneously?

Up to four cell populations can be sorted simultaneously, two sorted left and two sorted right. The remainder of cells are passed to waste. Each population can be identified with multiple parameters; i.e. multiple fluorescent probes, size and internal complexity. It is often useful to sort a negative (not the cells of interest) and positive populations (cells of interest) to have an internal control for whatever assay is performed on the sorted sample.

What concentration and in what medium should my cells be in?

Again this depends on your cell type. Some cells tend to clump at higher densities. In general, 5-10 X 106/ml in PBS or HBSS with 1-2% BSA will be fine. If your cells are happiest in something like RPMI, you should use RPMI that is phenol and biotin free (HyClone Laboratories). Cells should be brought in a Falcon 12x75mm tube (preferably polypropylene). (see Sort Buffer Selection and Ideal Cell Suspension Concentrations)

What should I bring for my sorted sample?

One to four 12x75mm Falcon polypropylene tubes with approx. 1ml of support media which contains 20% FBS (or some other source of protein such as BSA) and antibiotic (pen/strep or gentamicin). When sorting larger cells (i.e. using a larger nozzle size), the droplets sorted are larger and therefore generate a larger volume of sorted sample. In this case it may be necessary to bring two 15 ml conical tubes with 3mls support media to accommodate the larger volume. The same applies when the sorted sample contains more than 4x106 cells. You must also bring a completed and signed (by PI) Sort Sample Biosafety Form  for each sample to be sorted.

How should I treat my sorted sample to insure the best recovery and viability?

Cells can be a bit compromised by the trauma of the sort. The cells should be spun down as soon as possible following the sort, and re-suspended in their usual medium at a concentration that they are most happy and cultured under their usual conditions. Keep in mind that the condition of the cells prior to the sort will greatly impact their viability in the end. The healthier they are going into the sort the better the post sort viability.

What do I need to do prior to scheduling a sort?

You must first have an account opened with the facility by completing a Request for Flow Cytometry Core Access. You must also have a Project Biosafety Form on file. It is advisable to set up a consultation with Joanne Lannigan (924-0274; flowcytometry@virginia.edu) to discuss the specifics of your experiment prior to scheduling your sort. You must bring an original signed Sort Sample Biosafety Form with you for each sample to be sorted. Samples can not be sorted unless you bring this form with you, signed by the Principal Investigator.