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Investigators must read and comply with 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.

Contact the facility manager no later than 24 hours before the scheduled sort to confirm or cancel your appointment.

Review the sort logic and experimental design with the facility manager. Be prepared to supply 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 flouro-chrome combination
• downstream application for the sorted cells
• sterility requirements

Filter samples before sorting begins to remove any cell aggregates by running the sample through a sterile 40-80μm nylon mesh in a bio-safety cabinet.

Store samples in sterile-capped tubes (12x75mm snap-cap tube or 15ml conical tube) with proper labeling.

Provide collection tubes partially (1/4-1/3) filled with appropriate collection medium.

Select from of the following collection media:

• Culture media with at least 20% FBS
• PBS if collecting cells for RNA or DNA
• Fetal bovine serum only for very fragile cells

Re-suspend your cell sample in appropriate sorting buffer at the proper concentration:

• 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)

Resuspend cells in sorting buffer containing 10 units/ml DNAse during the sorting – this is for cells with high dead cell concentration, to avoid clumping of the cells in the sample by the released DNA.

Concentrate the samples to be sorted. Note that 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.

Prepare your sample in a concentration close to the values shown in the table below. You should bring extra sorting buffer in case if your sample is too concentrated.  Minimum concentration of the sample is 10 million cells per ml.

Include all the appropriate controls to set up the instrument for cell sorting. This includes:

• 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.

You should have a clear idea of the scattering profile and the fluorescent patterns of your target cells before starting the sorting. We recommend performing 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

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.