RRC-FCS: Sort Protocol

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We have two sorting flow cytometers: Beckman-Coulter EPICs Elite-ESP and the Dako-Cytomation MoFlo high speed sorter. Most of the instructions below apply to both sorters.

  1. Be sure to have some 5 ml round bottom tubes with strainer caps available (Falcon Tube #2235). Cells must be filtered just prior to sort! Filter your cells in your hood with these special tubes. The mesh on the caps of these tubes will NOT keep your cells sterile once the tubes are removed form the hood. Please change the strainer caps to regular sterile caps before transporting them to the flow lab if you want your cells to be sterile after sorting.

  2. Prepare your cells as you normally would for flow cytometry analysis. Cells to be sorted must be in a 5 ml round bottom tube (larger tubes can be used for the MoFlo) in PBS or serum-free culture media with a small amount of BSA (0.5% to 2%) and 2 mM EDTA to keep the cells viable and non-aggregated. The amount of BSA you use will vary according to the type of cells you are sorting. For the Beckman-Coulter Elite, the cell concentration should be approximately 2-4 million cells/ml (300µl - 500µl volume). If you want to sort more cells, then sorting will be performed using the Dako-Cyomation MoFlo high speed sorter. The concentration of cells to be sorted on the MoFlo should be 4-20 million cells/ml in a minimum volume of 400µl Please leave some extra PBS-BSA-EDTA buffer for us in case the cells are very sticky or you have underestimated the concentration of cells in the sample you wish to have sorted.

  3. When you give us the cells to be sorted, you must supply 5 ml round bottom tubes that are 3/4 full of medium with a high amount of protein. We recommend culture medium (with ANTIBIOTICS/FUNGIZONE) with 30% FCS. You must supply us with enough of these tubes (already filled) in order for us to complete your sort. If we have clogs or other problems, we will need more tubes so it is wise to leave us with extra tubes and medium.

  4. If you wish to sort dim positive cells, please supply us with a negative control so that we do not end up sorting negative cells for you.

  5. If you are using the Dako-Cytomation MoFlo high speed sorter, you may bring your samples in 1 ml Eppendorf, 5 ml, 15 ml, or 50 ml tubes. You must supply either 5 ml or 14 ml round bottom tubes with adequate collection media for your sorted cells.

  6. Please comment on any other special handling requirements for your samples, i.e. light sensitivity, tendency to clump, unstable out of incubator, keep on ice, etc.

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