Cell Sorting

Preparing for a Cell Sort

Successful sorting depends on both sample quality and planning with the operator. Please fill the sort form and check the Cell Sorting section in our Guidelines or further information. Unless the experiment is a pilot sort or a fluorescent-tagged cell enrichment, the staining panel and the population gating strategy must first be validated on an analyzer to ensure that the population(s) of interest can be sorted, and to avoid wasting resources, time and money. 

Sample preparation

  • Cells must be in a single-cell suspension, filtered through strainers.
  • Presence of debris, salt crystals and platelets must be reduced.
  • Recommended buffer: PBS with <3% serum ± EDTA (0.5-2 mM) (Phenol Red only allowed for sorts on the MoFlo ASTRIOS)
  • Only use antibodies/reagents required for the sort decisions (if you want further information about your sample, take an aliquot and stain it for that purpose).
  • Keep cells on ice to preserve viability.
  • Aim for the following concentrations:
    • 85 µm nozzle: 20-40 x 106 cells/ml
    • 100 µm nozzle: 10 x 106 cells/ml
    • Single cell sort: less than 5 x 106 cells/ml

Controls

  • Always bring appropriate controls depending on your staining (e.g., unstained, FMO, isotype, negative and positive controls). Please contact us if you need advice.
  • Provide single-stained controls for each fluorochrome when compensation or unmixing is needed.

Sorting process

  • Sort speed largely depends on cell quality, size, and abundance of target population. Rare populations take longer to sort (please see sorting times in the table below).
  • Purity typically reaches 95–99%, but viability and sterility depend on handling and setup
  • Post-sort purity check is highly recommended.
  • Sort into FACS tubes or appropriate collection tubes/plates containing medium/serum.
  • Clumps, debris, or poor sample preparation reduce yield.
  • In RNA extraction sorts: i) prefer live cells, ii) collect directly into medium/serum, RNA protection or extraction buffer, or single-cell plates with lysis buffer (no trizol), and iii) keep RNase-free conditions and consider adding RNase inhibitors.
           Frequency
Target number		 1 in 100,000 1 in 10,000 1 in 1,000 1% 10%
100 22 min 2 min < 1 min 1 sec < 1 sec
1,000 3.5 h 22 min 2 min < 1 min 1 sec
10,000 35 h 3.5 h 22 min 2 min < 1 min
100,000 15 days 35 h 3.5 h 22 min 2 min
1,000,000 150 days 15 days 35 h 3.5 h 22 min

Checklist: What to Bring for a Sort Appointment 

  • Cell samples in tightly capped tubes (no strainer caps!) prepared as single-cell suspensions, kept on ice, and concentrated in recommended buffer (see recommended cell concentrations above). 
  • Dilution buffer (PBS + <3% serum ± EDTA). 
  • Collection tubes or plates pre-filled, or ideally coated overnight in the fridge, with collection buffer/medium (correct volume depending on tube type and expected yield). 
  • Appropriate controls. 
  • Single-stained controls for each fluorochrome (or beads if cells are limiting). 
  • Viability dye (non-live permeable, DNA-intercalating dyes are highly recommended) or request one from the facility staff. 
  • FACS tubes with strainer caps for on-site sample filtering. 
  • Ice box with tightly closing lid for sample and collection tube transport, and storage during sort.