Cell sorting sample preparation

It is very important to read and comply to the following guidelines for preparing samples for cell sorting to avoid potential rejection of the sample.  Consult the core facility manager if you have any questions or concerns:  

 

1. Confirm your appointment or inform your cancellation with the facility manager no later than 24 hours before the scheduled sort.

 

2.  It is required for the researchers who need the cell sorting service to go over the sort logic and experimental design with the facility manager.  Please ensure you are available for this pre-sorting review and provide the facility manager the following information:

    preferred date/time of the sort

    cell type and cell origin

    approximate cell count in the sample and percentage of the target cell population(s)

    experimental design and flourochrome combination.

    downstream application  for the sorted cells

    sterility requirements

 

3.  Samples must be filtered to remove any cell aggregates by running the sample through a sterile 40-80μm nylon mesh in a biosafety cabinet before the start of the sorting. 

 

4.  Samples should be stored in sterile capped tube (12x75mm snap-cap tube or 15ml conical tube) with proper labeling.  Enough collection tubes (12x75mm snap-cap tube or 15ml conical tube) partially (1/4-1/3) filled with appropriate collection medium should be provided:


    Collection medium (choose one of the following):

   culture media with at least 20% FBS

   PBS if collecting cells for RNA or DNA

   Fetal Bovine Serum only for very fragile cells. 

 

5. Resuspend your cell sample in appropriate sorting buffer at the proper concentration:

   Sorting buffer:

   1x PBS (Ca/Mg2+free) 

   1% FBS or BSA

   25mM HEPES pH7.0

   1mM EDTA (optional, use it if sample cells are sticky such as DCs, granulocytes, fibroblast,        epithelial cells)

 

For cells with high dead cell concentration, to avoid clumping of the cells in the sample by the released DNA, resuspend cells in sorting buffer containing 10 units/ml DNAse during the sorting. 

 

Concentrate the samples to be sorted:
The sorting speed (and thus the length of the time for sorting a particular sample) will be determined by several factors including nozzle size (which depends on your cell size) and your sample concentration and total volume.  The larger the cells, the larger the nozzles to be used and thus the slower the sorting speed and the longer the sorting time.  The lower your sample concentration and the larger the total volume, the longer sorting time. 

 

It is crucial to prepare your sample to the correct concentration for the best sorting results:


sorting speed/nozzle size

cell type examples

sample concentration (cells/ml)

high speed/70μm nozzle

splenocytes, lymphocyte, mice bone marrow cells

30-40 million

medium speed/85μm nozzle

cell lines

20 million

low speed/100μm nozzle

DCs, macrophage, granulocytes

10 million


Have your sample ready in a concentration close to the above values.  Please bring some extra sorting buffer in case if your sample is too concentrated.  Minimum concentration of the sample is 10 million cells per ml. 

 

6. Please include all the appropriate controls to set up the instrument for cell sorting. You need to bring

    at least one negative (unstained and/or isotype; bring extra for initial setup if it is your first time sorting the sample) for each cell type/origin.

    one compensation sample for every fluorochome or dye that you use in your staining.  You can use your own cells, or ideally compensation beads, for compensation controls. 


Ensure your compensation controls have a distinct sharp clear positive peak that has no overlap with the negative peak. Control tubes should be at 1x106 cells per ml.

 

9. You should have a clear idea of the scattering profile and the fluorescent patterns of your target cells before starting the sorting.  We recommend perfoming flow cytometry analysis of your samples first.  Generally, the more discrete the target population from the unwanted cells, the better the purity and the yield of the sorting

 

10. Please allow for one to several times of test sorting to work out the optimal sorting conditions during the initial trial of sorting new cell samples.

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