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
• 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
media with at least 20% FBS
• PBS if
collecting cells for RNA or DNA
Bovine Serum only for very fragile cells.
5. Resuspend your cell sample in appropriate sorting buffer at the
1x PBS (Ca/Mg2+free)
1% FBS or BSA
(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
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
lymphocyte, mice bone marrow cells
medium speed/85μm nozzle
low speed/100μm nozzle
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
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