Sorting guideline
1. Fill out the online Service Request and Biosafety forms.These are required in order to set up the machine and to determine the appropriate biological safety protection. For first time users, please meet with the operator before the scheduled sample run to briefly discuss the overall nature of the experiment - design, appropriate controls, endpoint, etc.
2. Make a reservation online.
It is on a first-come-first-serve basis so it is advisable to make the reservation as early as possible. The availability of the machine can be checked on the public appointment calendar. For sorting, make the reservation at least two days in advance so the sterility of the machine can be checked.
3. Provide control samples.
The investigator/requestor is responsible for designing the experiment and providing the appropriate and necessary control samples - blank, untreated, untransfected, non-induced, isotype-labeled, etc. Enough cells as a blank control should also be provided to set the voltage, the initial gate, and the drop delay frequency - if sorting. 5 x 105 cells in 0.5-1ml are enough.
4. Properly prepared samples.
Samples should be aggregate-free, resuspended in serum-free medium and in 12 x 75mm 5ml polystyrene round-bottom tube (BD Falcon 352054) ready for loading. Aggregates can be removed by filtering samples through a 40-70um mesh or cell strainer. BD Falcon (352235) 5ml polystyrene tube is recommended because it has cell-strainer cap (70um) and the right tube size for loading in the flow cytometer. (Note: It is extremely important to filter the samples and remove the aggregates because they will clog the nozzle. Unclogging the nozzle and resetting it take significant downtime because the laser intercept, the point where the laser interrogates the cells, has to be realigned and the sort parameters have to be reset also if sorting.) 1-2 x 106 cells/ml is the optimal concentration for sorting at 1000-2000 events/sec. Minimum volume is 0.5ml. If the concentration is less than 106cells/ml, please let the operator know so the initial sort rate can be adjusted.
5. Provide collection tubes.
If sorting, provide 12 x 75mm 5ml tubes with 2ml of appropriate medium for the cells. Provide enough tubes to complete the whole run. It is recommended to increase both the serum and antibiotic concentration (if possible) two-fold in the medium in the collection tubes because it will be diluted with the sheath fluid (sterile 1X PBS) as the sorted sample is collected. Because the collection chamber is exposed to regular air (unlike biosafety hoods), there is potential for contamination.
6. BSL1 (Biosafety Level 1) and some pre-approved BSL2 samples only.
Because the flow cytometer fluidics is high pressure and the sample is sorted (deflected) out from jet-in-air, it generates aerosols. There is a great potential for airborne transmission, therefore potentially biohazardous materials such unfixed primary human cultures and established cancer cell lines are not accepted. Some BL2 samples such as lentivirus-infected mammalian cells will be accepted if pre-approved by the operator and/or the biosafety coordinator.
Biosafety guideline
1. CDC Biosafety Levels2. Biosafety Guidelines for Sorting Unfixed Cells
3. Updated Biosafety Guidelines
