Flow cytometry: rules for cell sorting

It is the user's responsibility to inform the Flow Cytometry Facility Staff about the source of the cellular material and and fill out biosafety information in the PPMS booking. Screened primary human material can be sorted on all FACSArias by a facility staff operator. Screened material will have negative test results for standard blood bank testing (HIV, Hepatitis B, Hepatitis C). Unscreened primary human material from patients or unknown sources can be sorted on the LEPH FACSAria.

To book your sorting experiment, please make a sort request in PPMS. You must complete a booking request form explaining experimental design (e.g. the fluorochromes being used, diagrams of the cell populations, amount of sorted material expected, and sorting regions desired). If you do not, your appointment may be rejected. 

Samples must be filtered through (40um mesh) before sorting. For example, you can use Falcon 2235 tubes with the mesh in the caps from Becton Dickinson. The optimal concentration of material in the sample tubes: (15-17) x10^6 cells /ml. Cells bigger than lymphocytes might need to be at lower concentrations. The following measures are important to prevent sorting material from clumping:

1. The concentration of fetal calf serum or BSA should not exceed 0.5%
2. Use a filtered buffer that is Ca2+ and Mg2+ free unless measurement depends on these ions
3. Add some 0.02% EDTA
4. Use anti-clumping agents, such as Accumax.

It is highly recommended that unstained and single-color stained controls be provided for optimum set-up.

Sufficient amount of collection tubes (1.5mL, 5mL or 15-mLcapacity) with media (up to 0.5mL or 3mL per tube, respectively) is necessary. To sort populations of low frequency, a higher volume of media is recommended for better cell recovery. Media in collection tubes should contain at least 10% serum to increase the viability of the cells. If many cells are expected to be collected, the serum concentration can be adjusted to 100% for dilution with the sheath fluid.

In case of delay or cancelation, notify cell sorter facility personnel as soon as possible by phone or e-mail: Ewa.Menet@yale.edu, Lesley.Devine@yale.edu, Jesse.Bonin@yale.edu, Zhao.Zhao@yale.edu, or Ryan.Duggan@yale.edu. We reserve the right to suspend the reservation privileges of individuals who repeatedly cancel with <24 hrs notice.

Charging policy

The charging policies described below are put in place to deliver the lowest priced services possible based on cost recovery. Machine "hoarding" (scheduling a large amount of time with no intention of using all of the scheduled time) and then canceling all or a portion of the time, is not fair to other researchers. This has been an issue in the past and slowed the pace of research on campus by preventing others from having access to machines. If this were this to go unchecked, it would result in higher fees for all investigators.

Sorting time will be charged based on the number of hours reserved or the number of hours actually used, whichever is greater. If someone reduces or cancels the previously booked time on short notice (less than 24 hrs) they will be billed for that time (similar to healthcare cancellation policies, due to the unlikelihood of filling that vacancy on short-notice). Sorting no-shows will be similarly charged, even in the case of accidental loss of material (cells died etc.). In the event that another investigator uses all or a portion of a cancelled time, the person with the original reservation will only be responsible for the remaining time not utilized.