Steps:
- Researchers, fellows, and external users who wish to start using the FACS service must contact Samantha Solito to discuss the use of the equipment before planning the experiment.
- Consult the FAQ and the FACS Aria III configuration.
- Provide details of any previous experiments, if applicable. It is strongly recommended to perform preliminary experiments to define staining and cell preparation procedures, rather than doing so during the final experiment.
- Sorting reservation with assistance; many factors (including sample preparation, the need for additional pre- and/or post-sorting cleaning, etc.) can influence the required time. We will decide together how much time to reserve to ensure there is enough time to sort your sample. The use of the BD FACSAria III will be scheduled through the Agendo system.
- At least two or three working days before the sorting experiment, contact the operator to discuss all the experiment details.
- At the time of the appointment, please be punctual and bring:
- Cell suspensions and controls in FACS tubes (5 mL round-bottom sterile polypropylene tubes with caps or other applicable collection devices), resuspended in the RESUSPENSION BUFFER. It is mandatory to filter samples immediately before sorting using cell strainers with pore sizes of 20–50 µm.
- Labelled collection tubes (5 mL round-bottom sterile polypropylene tubes with caps or other applicable collection devices) containing 1–2 mL (depending on the collection devices) of COLLECTION BUFFER.
- 10 to 50 mL of filtered (0.2 µm) resuspension buffer and collection buffer to dilute cells if necessary.
- Always check with the operator that all gates are set correctly and that the populations to be sorted are clearly identified.
Resuspension Buffer Composition:
- PBS
- 2% FBS or BSA
- Antibiotics
- 2 mM EDTA
Collection Buffer Composition:
- RPMI (or IMDM)
- 30% FBS
- Antibiotics
- 2 mM EDTA
- HEPES