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Flow Cytometry Core Laboraotry
User Guidelines
- To use the facility you must provide us with a responsible party's
name and mailing address. Accounts are billed monthly or bimonthly
according to the number of hours you use the machine(s). If you are
paying a flat yearly fee, that deduction will be made once yearly in
July (flat fees DO NOT every flow sorting or multicolor analysis on the
MoFlo - see fee schedule).
- 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 human
samples/pathogens available in the Core Lab.
- 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 (see
fee schedule for pricing). The MoFlo cell
sorter/5+ color analyzer is ONLY run by core employees and all experiments must be discussed with and
scheduled with a core facility employee in advance.
- The core is always available to consult on protocols and procedures
prior to implementation free of charge and we strongly recommend you come
to us before starting any new experiment so as to avoid any expensive
mistakes. Bear in mind that the facility at present has only 1
hands-on employee and is very busy with cell sorting, so if you need
help planning an experiment, please contact us well in advance
(sometimes 1 to 2 months ahead). 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 and 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 Dako Cytomation Moflo’s multi-color capabilities.
- All FACSCalibur data must be
removed from the FACSCalibur MAC on a Zip disk or portable flash or hard
drive. Please do not leave your data on the hard drive. 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 for advice on sample preparation.
- NO SAMPLES for CELL SORTING will be accepted without prior
consultation with a core employee.
- All samples to be run on the Becton Dickinson equipment must be in 12
X 75mm POLYSTYRENE tubes ONLY FROM FALCON. Samples to be run on the MoFlo
must be in 12 X 75mm POLYPROPYLENE tubes
(click here for info
on
ordering 12 X 75 tubes).
- ALWAYS bring NEGATIVE AND POSITIVE CONTROLS. To 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 or 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. Core employees cannot be held responsible
for any experiment they are assisting on if the investigator does not
bring negative and single color positive control tubes.
- All samples must be filtered immediately prior to running (this is for
analyzers or sorters) 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 #35 2340 - 40um and Falcon#35 2350 – 70um) and in filter top
caps found on Falcon brand polystyrene 12X75 mm tubes (Falcon #35 2235).
It is also available in sheets from Sefar America Inc. We also recommend samples
should be kept on ice or chilled until ready to run on the machine.
Further, samples utilizing PI for DNA ploidy/cell cycle analysis
should be mixed vigorously with a TB syringe, filtered with nylon mesh and
then 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.
- All users are responsible for daily maintenance of the machine. After
use, every user should follow the
FACSCalibur
Start-up and End-of-Run Maintenance 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 FACStations (Mac
computers that will run Becton Dickinson software) when not already booked
for acquisition and several other stand-alone FACStations on the 2nd floor
of the CA building. These computers have Becton Dickinson CellQuest 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 FloJo and 2 Mac's
running OS10 Jaguar have CellQuest Pro (Becton Dickinson's newest
version of CellQuest). Modfit is also available on the FACStations for
DNA ploidy and cell cycle analysis. There are a
few free analysis software programs available on the online (i.e.
Summit from DakoCytomation, WinMDI) .
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