Frequently Asked Questions (FAQ)

Sample Preparation for Cell Sorting

  1. What cell concentrations should the samples be at prior to cell sorting?
    • The maximum cell concentration for samples differs between instruments. Please refer below for a guideline on the maximum number of cells per mL for the following instruments.
    • For BD FACSAria Sorters
      • Sample concentration:
        • Peripheral blood mononuclear cells (PBMCs): 10 million cells per mL
        • Primary cell lines/tissues: 5 million cells per mL
    • For BD Influx Sorter
      • Sample concentration:
        • Peripheral blood mononuclear cells (PBMCs): 7 million cells per mL
        • Primary cell line/tissues: 5 million cells per mL
    • For MoFlo XDP Sorter
      • Sample concentration:
        • Primary cell line/tissues: 15 million cells per mL
  2. What are key points to look out for during sample preparation?
    • Before preparing your samples, consider the following:
    • Cell Viability?
      • Stain your cells with a viability marker, i.e. Propidium Iodide (PI) or 4′,6-diamidino-2-phenylindole (DAPI); to provide data on the health of your samples
    • How to ensure samples are in single-cell suspension?
      • Re-suspend cells in a buffer containing EDTA or DNase
      • Filter the samples through a 70µM filter paper right before sorting
    • For cell sorting, what is your downstream experiments?
      • For culture, use complete media with antibiotics
      • For genome sequencing, cells can be sorted directly into lysis buffers
  3. What are the types of sample collection devices recommended for cell sorting
    • For BD FACSAria™ and MoFlo XDP Sorters
      • Sample tube: Polystyrene and Polypropylene
        • Tube sizes: 5mL tubes and 15mL Conical tubes
      • Collection tube: Polypropylene
        • Tube sizes: 1.5mL microtubes, 5mL tubes and 15mL Conical tubes
        • Plates/PCR: 6 to 384 well-plates, PCR tubes/strip/plates
    • For BD Influx™ Sorters
      • Sample tube: Polypropylene
        • Tube sizes: 5mL tubes
      • Collection tube: Polypropylene
        • Tube sizes: 1.5mL microtubes, 5mL tubes and 15mL and 50mL Conical tubes
        • Plates/PCR: 6 to 384 well-plates, PCR tubes/strip/plates
  4. What do you need to prepare for cell acquisition?
    • Prepare sample in 5mL Polystyrene tubes
    • Cell concentration: 2 million cells in 200uL of FACS buffer

Things to note:

  • Please be on time for your booking slots.
  • Prepare samples in appropriate tubes according to the instrument of use.
  • Ensure collection tubes/plates contain media with antibiotics or FACS Buffer.
  • If the sample is too concentrated, please dilute with FACS Buffer.
  • All samples must be kept in a closed-lid icebox containing sufficient ice during users’ transportation of samples to the SIgN Flow Cytometry platform facility. This is to maintain cell viability and prevent photo-bleaching of fluorochromes.
  • Filter the samples through a 70µM filter paper before sorting and acquisition.
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