Center for Molecular Medicine Cologne

Road map for cell sorting services

1. First visit

  • Make an appointment with the Facility staff (preferentially by email).
  • Aim of first visit is to define the sort procedure and the experimental prerequisites if necessary. Definition includes marker stainings, optimized work flow, sort procedure time, date of pre-run and date of sort.
  • Bring with you a complete list of the probes which you want to analyze or to sort. Staining data and flow analyses of targets you want to use for sort are welcome.
  • Classify the biosafety levels of your probes using the "safety data sheet".

    2. Pre-run

    • Fix date of pre-run with the Facility staff (by email or phone). Announce any delay immediately. "No show" costs equivalent to 1 hr pre-run analysis.
    • Prepare test samples with your cells for analytic pre-run. ALWAYS bring with you APPROPRIATE CONTROLS in sufficient cell numbers (1x 105 - 1 x 106 cells). Controls help to gate cells of interest in an optimized fashion. Negative controls are cells stained with an isotype matched control antibody. Specificity control in an indirect staining procedure is staining with the secondary antibody only in order to indicate "non-specific staining" or Fc binding of the secondary antibody. Compensation controls are required for staining using two or more fluorochromes and stainings with each dye separately.
    • Bring with you filled form "Safety data sheet" and the Account form ("Service")
    • Facility staff will run basic acquisition setup, compensation, gating for antigen(s) of interest for sort. Data analysis of your samples will be discussed and staining adjusted if necessary.
    • NO SAMPLE will be accepted without pre-run analysis.

    3. Cell sorting

    • Fix date for sort with the Facility staff (by email or phone). Announce any delay immediately. "No show" costs equivalent to 1 hr sort run.
    • Prepare your samples and perform staining using the protocol and procedure as performed in the pre-run analysis.
    • Bring with you an fresh, unused CD-ROM/DVD for data transfer.
    • All samples run on the BD Biosciences instruments must be in 12 x 75mm polystyrene tubes (BD Biosciences). If not otherwise defined in the pre-run, cells should be 1×106 to 1×107/ml for the FACS Aria, and each tube should have a volume adapted to the cell counts of the sample.
    • To avoid cell clumps, cell buffer may contain 2mM EDTA if not otherwise defined in the pre-run.
    • All samples run on the Miltenyi Biotec instruments must be in 15ml polystyrene tubes (BD Biosciences). If not otherwise defined cells should be 2×108/ml for the AutoMACS Classic and AutoMACSPro.
    • To avoid cell clumps, labeling buffer may contain 2mM EDTA, or alternatively use AutoMACS "Running buffer" (Miltenyi Biotec) for magnetic labeling of cells.
    • For sorting on the FACS Aria instrument, ALWAYS bring with you APPROPRIATE CONTROLS in sufficient cell numbers (1x 105 - 1 x 106 cells). Controls help to gate cells of interest in an optimized fashion. Negative controls are cells stained with an isotype matched control antibody. Specificity control in an indirect staining procedure is staining with the secondary antibody only in order to indicate "non-specific staining" or Fc binding of the secondary antibody. Compensation controls are stainings with each dye separately.
    • Samples should be kept on ice or chilled until ready to run.
    • Bring with you enough sampling tubes for sorting (BD FACS Tubes with 500 μl of cell culture medium).
    • All samples will be filtered by the Operator shortly before start of sort run.

      GentleMACS Tissue Dissociation

      • Fix date for tissue dissociation run with the Facility staff (by email or phone). Announce any delay immediately. "No show" costs equivalent to 1 hr GentleMACS run.
      • A first visit before run to discuss the procedure and sample preparation is strongly recommended.
      • GentleMACS protocols are available from Miltenyi Biotec.
      • User must bring the samples in C-Tubes (for single cell suspensions) or M-Tubes (for RNA and DNA isolation).
      • Instrument will be run by the Operator only.

        Magnetic cell sorting

        • Fix date for sort with the Facility staff (by email or phone). Announce any delay immediately. "No show" costs equivalent to 1 hr AutoMACS run.
        • A first visit before run to discuss the procedure and sample preparation is strongly recommended.
        • Labelling of cells for magnetic cell sorting are done by the user according to recommended protocols.
        • Labeling protocols are available at www.miltenyibiotec.com. Modifications may be done if required.
        • Magnetic sorts are run by the Operator.
        • All samples are filtered through a cell strainer (30 μm) immediately before sort to avoid cell clumping during magnetic cell separation