Download Aria Sample Prep 110210

Sample Preparation for the CHORI FACSAria
So that we may choose the proper nozzle to use for sorting, please determine
the diameter (in suspension) of the cells you wish to sort or bring us a sample
before you sort so that we can size the cells.
It is imperative that you notify us before the sort if your sample contains any
infectious agent, whether pathogenic or nonpathogenic, human or nonhuman
host. Infectious agents would include viruses, bacteriophage, bacteria, fungi,
parasites, etc. Please notify us also if you will be sorting a cell line that has
undergone viral transformation, e.g., with Epstein-Barr virus.
The following tubes should be brought to the sort:
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Unstained cells
Compensation tubes containing singly-stained cells for each dye to be
used in the study. Ideally, about 50% of the cells in the tube will be
positively stained. The marker used for the compensation tube need
not be identical to the marker used in the sample tubes, as long as the
positive cells are as bright or brighter than the cells in the sample
tubes. However, the fluorophore must be identical to that used in the
sample tubes. If a sufficient number of positively stained cells cannot
be achieved for proper compensation, the use of BD CompBeads is
highly recommended. These beads capture the antibody of interest
and come in three different preparations – 1) Anti-mouse Ig, ! (BD
552843), 2) Anti-rat Ig, ! (BD 552844 ), and 3) Anti-rat/hamster Ig, !
(BD 552845).
Area scaling tubes. If compensation tubes are prepared with the cells
to be sorted (as opposed to beads), then they should also be suitable
for area scaling. For single color experiments, for which compensation
tubes are unnecessary, or if beads are used for compensation, then
separate tubes will be needed for area scaling (making certain the
digital signals for peak area and height are comparable). Area scaling
tubes should contain the same cells that are to be sorted. Ideally, at
least 50% of the cells in the tube will be positively stained. One area
scaling tube will be needed for each laser required to excite the stains
used for your experiment. If you are unsure which laser is used to
excite a particular dye, please consult us before you set up your
experiment.
Sample tubes. The ideal cell concentration for sorting is 5 " 106 – 10 "
106 cells per ml. If possible, cells should be suspended in buffer or
medium that does not contain phenol red (which autofluoresces). If
there is a tendency for the cells to clump, measures should be taken to
prevent cells from aggregating during the sort (e.g., addition of EDTA
to 1 mM in sample buffer). Protein (BSA or fetal bovine serum) should
be kept to the minimum necessary to ensure cell viability, as the
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presence of protein diminishes resolution of cell populations during
sorting. Because cells are diluted in sheath fluid as they are sorted,
addition of HEPES to a final concentration of 25 mM in sample buffer
will help maintain proper pH and enhance cell survival. Cells should
be filtered through 40 !m (or narrower) mesh to remove clumps after
staining is complete and the final cell suspension is prepared. We can
provide BD Falcon 12 " 75 mm 35 !m filter-top tubes for this purpose
at a charge of $1.00 per tube.
The sample injection chamber has a heating/cooling unit that can
maintain the sample temperature at 4°, 20°, 37°, or 42° C. The
heating/cooling unit is meant only to maintain the sample temperature,
not to cool or heat the sample. If you wish to use this option during
your sort, please let us know before your sort and bring your sample(s)
to the sorter precooled or preheated to the desired temperature.
Collection tubes or plates. Polypropylene tubes are preferred as cells
are less likely to adhere to the walls of these tubes. Tubes can also be
precoated with a BSA solution to prevent sticking to the side of the
tube. Cells can be collected into up to four 1 ml microtubes (Bio-Rad
no. 223-9391, which we can supply), up to four 12 " 75 mm tubes, up
to two 15 ml conical-bottom tubes, into multiwell plates, or onto slides.
The collection buffer or medium of your choice should be added to
each collection tube in the following volumes: 1 ml tube: " 100 – 200
!l, 12 " 75 mm tube: " 0.5 – 1.0 ml, 15 ml tube: " 4 –5 ml, plates:
variable, depending on number of wells, but bottom of well should be
completely covered. Collected cells can also be maintained at
temperatures other than ambient temperature by using special tube
holders connected to a circulating water bath. Please advise us ahead
of time if you wish to use this option.
Only cells that are to be sorted need to be filtered through 40 !m
mesh. Cells in setup and compensation tubes need not be filtered.
However, the sample buffer in which cells are suspended should be
0.2 !m-filtered. This applies to all tubes.
If you wish to have cells sorted under aseptic conditions, please advise
us of this when you first contact us regarding your sort. Aseptic sorts
require an overnight prep of the Aria fluidics with 70% ethanol.