Cancer Institute

  • Flow Cytometry Sample Preparation

    Each investigator is responsible for sample preparation for both the Gallios analyzer and the MoFlo XDP cell sorter. Please make sure that the samples are prepared as per standard requirement of the instruments. This will ensure good quality data. A poorly prepared sample runs the risk of clogging the instruments. Please contact us if you have questions regarding sample preparation.

    IRB approval, Biosafety

    Each PI is responsible for having current IRB and biosafety approvals.

    For investigators running their own samples

    • Keys

      Each investigator will be provided with a key to access the core facility for after hour’s use for the particular day. We request you to not pass on the key to other investigators and return it to the Facility Operator the next day. In case the key is lost, it will be the responsibility of the investigator to replace the lock to the Core facility at his/her own expense.

    • Instrument care

      It is the responsibility of the investigator to take the necessary care of the Gallios analyzer as discussed during the training. This will ensure smooth running of the Core facility.

      Please refrain from handling/touching the MoFlo XDP cell sorter in the absence of facility staff.

    Sample preparation for flow cytometry analysis (Gallios)

    For flow cytometry analysis the minimal volume of sample required is 0.5 ml; the optimal concentration is between 0.5-1 x 106 cells per sample. Please bring the resuspended cells in Falcon 5 ml 12 x 75mm polystyrene round-bottom tubes (BD Falcon cat. number 352054). Samples must be in a single cell suspension. If they are not, you run the risk of clogging the instrument since the tubing and nozzle through which the cells pass is very narrow.

    Basically, if you see clumps or aggregates of cells and/or debris in your sample, it will not run properly. Adding 1mM EDTA should prevent the formation of cell clumps. In order to remove clumps or aggregates the sample should be filtered prior to running. We recommend using Falcon 5 ml polystyrene round-bottom tubes with cell strainer cap (BD Falcon cat. number 352235).

    Sample preparation for cell sorting (MoFlo XDP)

    For sorting, a minimum of 10 x 106 cells is usually required; the optimal concentration is between 3-5 x 106 cells/ml. Cells ready for sorting should be resuspended and then collected in the media they are "happiest" in, which includes either commercially available FACS buffers, PBS, or cell culture media such as RPMI or DMEM. Please realize, that the presence of phenol-red in the culture media may affect the background florescence slightly, however in general this is not a problem. In the case of adherent or inherently sticky cells, it will help to add 1mM EDTA to the sorting buffer. Adding EDTA should prevent the formation of cell clumps, which will be aborted by the machine and not sorted. Also, addition of a buffering agent like HEPES (pH 7.0) to the sorting and collection buffers will help buffer the pH of the solution and aid in cell viability as the cells exit the sorter. Please bring the collection buffer of choice as well as extra sorting buffer in case the cells need to be diluted.

    Basic sorting buffer

    • 1X PBS or RPMI
    • 1mM EDTA
    • 25mM HEPES pH 7.0
    • 0.5-2% BSA or FBS

    Importantly the presence of dead cells and/or debris in the sample greatly reduces almost all the aspects of the sort, including efficiency, purity, accuracy and yield. This is in part due to the fact that 1) dead cells have higher auto florescence compared to live/healthy cells and will obstruct the detection of dim fluorescing populations, 2) dead cells are more subjective to non-specific binding of antibodies and dyes resulting in inaccurate selection and sorting of false positives, which affects the purity of the sort, 3) the presence of free DNA, released from dead cells in the sample leads to cell clumps and clogs, which can interrupt an jeopardize the entire sort. Also, data from clumped cells is aborted and the cells are discarded by the machine. A higher percentage of cells in clumps reduces the yield.

    Samples with large amounts of dead cells and/or debris must be cleaned before use. Depending on the specific problem, this can be done by either filtering the sample prior to sorting and/or adding DNAse to the sorting buffer. If the sample cannot be cleaned, it might help to do a viability stain using Propidium Iodide to discriminate live from dead cells and subtract the dead cells from the population. While the addition of Propidium Iodide will help in the resolution of the sort, realize that it adds an additional parameter and its use must be taken into account when selecting fluorochromes.