Planning your first cell sorting experiment

Steps:

  1. Fill out and submit the Account Registration Form.
     
  2. Fill out the Biosafety questionnaire. The questionnaire has to be signed by the PI and by the investigator who brings samples for sorting. Please send an electronic copy of the signed questionnaire to flowcytometry [at] email.chop.edu. The staff will send you log in info for the scheduler.
     
  3. Log in at   http://cytochop.bookedscheduler.com   and reserve a sorter.
    • Select the type of service from the drop-down list: Full service, Semi-assisted or Unassisted.
    • MoFlo Astrios users may select the nozzle size: "70 um (high-speed)" or "100 um (standard speed)" from the drop-down list. Most sorts on MoFlo Astrios are performed with 100 um nozzle.
    • For all high-speed sorts on MoFlo Astrios 30 additional minutes should be reserved for replacing the nozzle. Check with the staff if you are not sure how much time is needed for your sort.
    • The Biosorter has to be used for large cells (hepatocytes, adipocytes, etc.), cell clusters and small organisms.
  4. Bring to the Flow Lab in ARC 1207 the items listed below at the time of your appointment:
    • cell suspensions , including single color compensation controls, unstained cells and FMO controls if needed;
    • labeled collection tubes containing cell culture medium or FBS;
    • approx. 10 ml of buffer for diluting the cells if needed.
  5. Always check with the operator of the sorter to make sure that all gates are properly set and populations to be sorted are clearly identified.


For a good introduction to cell sorting watch The Art of Sorting webinar by Beckman-Coulter.

Some more details...

High speed vs. low speed sorting

Both MoFlo Astrios and FACSJazz are jet-in-air sorters. MoFlo Astrios can be operated in high speed mode (70μm nozzle) or in low speed mode (100μm nozzle). Only small cells with diameter smaller than 14 um (e.g. lymphocytes) can withstand the high pressure (60 psi) applied to samples during high speed sorting. Cells with diameters below 20 μm can be sorted at low speed (at pressures between 20 and 30 psi). Generally, the diameter of the cells to be sorted should be at least 5 fold smaller than the nozzle size. Note that FACSJazz can be operated in low speed mode only (100μm nozzle; 27 psi).

Sample preparation

Adjust the concentration of the cells to be sorted to maximum 20 million/ml for low speed mode and up to 30 million/ml for high speed sorting mode. We can dilute the cells if they are too concentrated, but for practical reasons we will not concentrate them. In principle, any physiologic buffer may be used to resuspend the cells to be sorted. It is essential to have the cells well dissociated and to prevent clumping in order to achieve good recovery rates and to prevent clogging of the nozzle. We recommend suspending the cells in Ca2+/Mg2+-free buffer, such as PBS (phosphate buffered saline) or HBSS (Hank's balanced salt solution) with low protein content (≤ 2% BSA).

EDTA (0.5 to 5 mM) and/or DNase (20 - 100 μg/ml) may be added to further prevent cell clumping. Note that standard cell culture media contain Ca2+, Mg2+ and proteins from serum that favor cell clumping. For pH-sensitive cells HEPES (25 mM) should be added to the buffer.
It is highly recommended to pass the cells through a cell strainer (BD Biosciences cat. 352235) immediately before sorting.

Collection tubes

On MoFlo Astrios up to 6 populations can be sorted in one pass (6 way sorting) in FACS tubes (5 ml) and up to 2 populations can be sorted in 15 ml conical tubes.
On FACSJazz maximum two cell populations can be sorted in one pass in FACS tubes or 15 ml tubes.
Please bring labeled collection tubes with approximately 2 ml of cell culture medium. FBS 100% may be added in collection tubes to help keep the viability rates of sorted cells high.

Both Astrios and FACSJazz have a movable stage and software support for collecting single cells in multi-well plates, including 96 well plates and high density plates (384 well plates).

Sorting time

Sorting time depends mainly on how many cells need to be analyzed and sorted. MoFlo can process up to 100 million events/hour in high speed mode. FACSJazz and MoFlo Astrios in low speed mode can process up to 36 million events/hour.
For diluted samples, the volume is also a factor that should be considered when estimating the time needed for sorting: maximum 4 - 5 ml of sample can be processed in one hour.

Cell sorting training

End-users who need to sort at least once a week are encouraged to learn how to operate a sorter of their choice. Basic cell sorting skills on FACSJazz or MoFlo Astrios can be gained in two training sessions of approximately 2 hours each. Typically it takes only one session of about two hours to learn how to use the Biosorter at basic level. Additional training may be needed for learning advanced features and for learning how to troubleshoot common technical problems.

For additional details call the Flow Lab at 215-590-3402 or send an email message to flowcytometry [at] chop.edu.



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