Campus Flow Cytometry Core Facility User Guidelines
To use the facility you must provide us with a responsible party's name (PI), charge
account # (CFC or other), email address, campus phone number and mailing address.
Accounts are billed monthly according to the number of hours you use the machine(s).
ONLY FIXED human cells are to be run in the Core Lab. You cannot run unfixed human samples or any unfixed animal samples that may be a biohazard to humans. Currently
there is NO sorting of unfixed primary human/non-human primate samples/pathogens available in the Campus Flow Cytometry Core Facility. Commercially
acquired Human Cell Lines may be sorted if approved by the AU Biological Safety Office.
All new users will go through a brief orientation before beginning use of the facility.
Training is provided for use of the analyzers but users may also opt to have samples
run by a core laboratory employee as time permits (see fee schedule for pricing). The MoFlo XDP 7-color Cell Sorter (& Analyzer) is ONLY run by core employees and
all experiments must be discussed with and scheduled with a core facility employee
The core is available to consult on protocols and procedures prior to implementation
and we strongly recommend you come to us before starting any new experiment so as
to avoid any expensive mistakes. Consultation/advice for non core facility cytometers
is available as time permits at the normal hourly assisted rate. The facility has
only 1 hands-on employee, so if you need help planning an experiment or acquiring
samples on the FACSCalibur, please contact us well in advance. You should ALWAYS prepare small test runs to make sure things are working before
scaling up any experiment.
Any non-routine multicolor/multi-fluorochrome experiment (i.e., not familiar to you/you have not done before) you wish to do SHOULD have a
pilot experiment done beforehand. In our experience, although our instrumentation
has four and five plus multicolor capability the antibody titering, instrument set-up
and compensation for three plus fluorochromes is not trivial and may take several
pilot runs to work out. It is imperative that you discuss any complex multicolor experiment
with a core employee prior to planning and/or ordering supplies so as to avoid any
problems or misunderstandings, this is even more important if you wish to utilize
the MoFlo's multi-color capabilities.
You should always take a copy of FACSCalibur data files with you after each acquisition
run. When you have amassed enough data on the FACSCalibur MAC's to fill a CD or DVD,
it is advised that you make 2 copies and take them with you to safeguard your data
in the event of a FACSCalibur computer failure. The Flow Cytometry Facility is NOT responsible for lost data, so please make sure that you have saved your data properly.
At various intervals all data on the hard disk will be removed.
Users must prepare their samples in their work areas. Core employees are available
(as time permits) for advice on cytometry sample preparation.
NO SAMPLES for CELL SORTING will be accepted without prior consultation with a core
ALWAYS bring NEGATIVE AND POSITIVE CONTROLS. To properly setup the cytometer and make appropriate conclusions about your samples
the proper controls are necessary.
If you have samples stained with MORE THAN ONE fluorochrome per tube then we recommend
you bring samples stained with each dye individually “SINGLE COLOR TUBES” (as well
as mixed experiment tube or tubes) and 1 tube of unstained/negative control cells
to allow for proper compensation. If you are staining for rare markers, then for every
dye that has a rare marker stain on it, you should also bring a single color staining
of your cells using the same dye as the rare marker antibody that is conjugated to
an antibody that will have a significant percent positivity for your cells or utilize
"Compensation Beads" that will bind your experimental antibodies to act as positive
controls; these beads are available from several flow cytometry reagent suppliers
(BD Biosciences, Life Technologies, eBioscience, Miltenyi Biotec). When using "Compensation Beads" with your antibodies for your positive control
tubes, you should also make a negative control tube using your unstained experimental
sample cells. Core employees WILL NOT be held responsible for any experiment they are assisting on if the investigator
does not bring appropriate negative and single color positive control tubes.
All samples must be filtered immediately prior to running (this is for analyzers and
the sorter) to avoid clogging the machines. Any excessive clogging of the FACSCalibur
by any one lab will result in charges for unclogging after the 3rd incident ($10/incident).
Thirty to seventy micron nylon mesh should be used. This mesh is available in Falcon
brand cell strainers for 50ml conical tubes (Falcon #352340 - 40um and Falcon#352350
- 70um) and in filter top caps found on Falcon brand polystyrene 12X75 mm tubes (Falcon
#352235). We also recommend samples should be kept on ice or chilled until ready to
run on the machine. Further, samples for DNA ploidy/cell cycle analysis may be mixed
vigorously with a pipette and then should be filtered with nylon mesh and kept on
ice immediately prior to acquiring on the machine.
PLEASE SHOW UP ON TIME. You should give at least 24 hours notice if you need to cancel
an appointment. Appointments made with a Core employee that are not cancelled by 11AM
of the prior day will be charged in full. If you are late for an appointment and run
into the next users appointment you must stop acquiring your samples and finish at
a later time. In particular, if you are going to be late for a sorting appointment
more than one hour, it is your obligation to check to see if your sort can still be
done because other sorts may be scheduled on the same day. If there are other sorts
scheduled, they will have priority and time after 5PM will be charged double.
All users are responsible for daily maintenance of the machine. After use, every user
should follow the FACSCalibur Start-up and End-of-Run procedure, that way, when it's your time, the machine should be ready for you to use if the
previous user has done their part. Users that do not follow the rules, for example
running unfiltered cells that clump and clog the machine, leaving the machine on,
not doing End-of-Run maintenance and making sure that the waste is emptied and the
sheath tank is filled, etc. will be identified and excluded from using the instruments.
These instruments are common equipment and have to be treated with respect so that
we may operate them for a long time without incurring additional expense due to misuse.
Data analysis is available on all FACSCalibur instrument FACStations (Mac computers
that will run Becton Dickinson software) when not already booked or in use for acquisition.
These computers have Becton Dickinson CellQuest Pro for acquisition and analysis.
Data analysis is also available on several other stand-alone FACStations on the 2nd
floor of the CA building; some of the stand-alone computers have Treestar's FlowJo in addition to CellQuest Pro (Becton Dickinson's newest version of CellQuest) and
Verity Software House's Modfit DNA Cell Cycle software. There are a few free analysis software programs available online (i.e. MFI, WinMDI). Cytospec is available from the Purdue Cytometry website and one that is free to academic users
is Flowing Software.