Identified interesting populations that now need isolation? You've landed on the right page!

How do I request a sort?

Link here!

What concentration do I bring my cells in?

Generally start at about 10-20 million cells per ml depending on how large & prone to clumping your cells are. We can always dilute cells if necessary so always bring some sterile (if needed) buffer. If you only have a tiny amount of cells, A minumum volume of 200-300ul is recommended.

How long will my sort take?

There is no generic answer. The frequency of the population, purity requirement, and operating frequency (how many droplets per seconds are generated will dictate how long the sort will take. 

The following can be used as a guide on the instruments here.This can be used to help plan accordingly how and when you will collect your cells from the sorters, how many cells may be an optimal number to sort as well as which collection media might be optimal to use. 

*80% efficiency

Sort Collection Consideration

Selecting the optimal collection adapter for the application is important!

We frequently receive questions regarding sort/collection tubes and what should be in them for cell sorting. I have prepared the below as a basic guide to frequently used sort/collection tubes buffers.

HEPES has been used in other flow laboratories, with success, to alleviate the pH changes that can occur in sort/collection tubes. PH changes can not only occur in the samples to be sorted (that can be kept at pressures upwards of 5-70psi for extended periods) but also in collection tubes. PBS/media containing carbonated based pH buffers are prone to pH change when not in a 5% CO2 environment such as an incubator.

We now have sterile 1M HEPES aliquots in the cell sorting labs that can be added to the sort/collection tubes upon request. As an example, to make a 20mM final concentration, 20uL needs to be added, per mL of sample, from WRHFlow 1M stocks. It is okay to have both bicarbonate and HEPES based buffering systems concurrently in samples.

Note: It is important that not only your cells, but your downstream application is compatible with your selected buffer protocol and that you consider how changing sort buffers by adding HEPES may impact previous/future data generation before implementing any changes. Some common application examples that can require specific buffers include annexin V staining, the calcium flux assay, nuclei sorting, single cell sorts and RNA/DNA extractions.

What can I bring my sample in?

Vessel:

AriaIII cell sorter – accepts Eppendorf tubes, 5/15mL falcon/FACS tubes as inputs.

Influx cell sorter – accepts 5mL polypropylene FACS tubes as inputs.

Buffer:

Cells to be sorted in

(1-5% FCS/FBS OR 1-2.5% BSA)

in

(PBS Ca/Mg++ free OR HBSS OR media, preferred without pH indicator)

recommended with

(10-25mM HEPES)

with/out, by requirement

(1-5mM EDTA OR DNase + MgCl₂ OR custom buffer)

What to collect my samples into?

Vessel:

AriaIII or Influx – 96 well, 384 well, Eppendorf tubes, 5/15/50mL falcon tubes

Buffer:

Appropriate collection vessel coated with or containing

(100% FCS/FBS – with a final concentration after sorting cells of >10% (recommended with 10-30mM HEPES – with a final concentration after sorting of cells >5mM & <30mM HEPES) OR cell culture media OR RNA/DNA extraction specific buffer OR custom buffer)

, 

How much sample can I collect per collection vessel?

This depends on the starting sample, sort setup and instrument frequency used. 

An approximate small guide (still in progress)

Nozzle Diameter Guide

• Nozzle size should be >4-6 times the size of the largest cell
• Nozzle size contributes to sort stability
• Larger nozzle normally mean lower sheath pressure
  • Less shear stress on cell
  • Less acceleration
  • Ideal for minimising dissolving gases
  • Liquid per droplet - final cell:sheath:collection buffer ratio
  • Lower frequency of droplet generation = <events per second
• Selecting nozzle diameter is not strictly as defined in the below table. This should be used as a guide only. 

Sorting for DNA extraction

When sorting volume is high, it will be necessary to sort into ice cold media or PBS and spin down your cells.  DNA is fairly robust and storage at -70^oC can preserve DNA quality.

When sorting volume is low, the best way is to sort directly into lysis buffer, either in-house made or from an extract kit.

Example of in-house lysis buffer recipe: 

• 50mM Tris-HCl, pH 8
• 1mM EDTA
• 0.5% SDS
• 50 ug/ml proteinase K
• 1x PCR buffer

After sorting, incubate at 56^oC for 40 min to digest proteins, and inactivate at 95^oC for 8 min. Mixture is now ready as DNA template for PCR.

You can have as little as 10ul in a 96 well plate for single cell PCR, or ~100ul for a few thousand of cells.

Sorting for RNA extraction

Please ensure you use RNAse free reagents and equipment. This include all buffers, tips and tubes. 

Viability stain is highly recommended, as dead cells are a large contributor of RNases.

When sorting volume is high, it will be necessary to sort into ice cold media or PBS and spin down your cells.  Remove supernatant and extract RNA as soon as possible. Taking the Cells-to-Signal kit as an example, 100ul of lysis buffer can handle up to 5 x 10⁴ cells in 20ul of residual volume.

If necessary, stain up some cells, as your protocol for extraction, and run a RNA quality check.

Having RNase inhibitor in collection buffer is helpful, but may not be economically feasible.

If you'd like to store samples long term, sort/re-suspend cells in 5-10 times volume of RNAlater. Once in RNAlater, samples can be stored for up to 1 day at 37°C, 1 week at 25°C, 1 month or more at 4°C, and long-term at -20°C or -80°.

When sorting volume is low, the best way is to sort directly into lysis / trizol buffer from your RNA extraction kit.

• Trizol can handle ~10% sample dilution, and Trizol LS can handle ~30% sample dilution.
• For RNA extraction kits, follow the ratio of lysis buffer:sample in the protocol (For example Qiagen RLT buffer is 100ul cells:350ul RLT buffer) 

Freezing cell pellet for storage is OK, but freezing cells in trisol/lysis buffer can have a big reduction on your RNA recovery.

Sorting fixed cells for RNA extraction

Zinc fixation is recommended. It preserves integrity/quality of RNA better than fixing with PFA, EtOH or commercial kits.

For a detailed protocol refer to this paper: Zinc Fixation for Flow Cytometry Analysis of Intracellular and Surface Epitopes, DNA Content, and Cell Proliferation.

Please ensure you use RNAse free equipments. This include all buffers, tips and tubes. If necessary, stain up some cells as your protocol for extraction and run a RNA quality check.

Viability stain is highly recommended, as dead cells are a large contributor of RNases.

Have 1:100 RNase inhibitor in all your buffers. Use 1:25 RNase inhibitor during your stain and final re-suspension.

Keep all steps on ice apart from fix & perm. You may require longer incubation time depending on how well your antibody stains.

For a more detailed protocol refer to this paper: Method for analyzing RNA following intracellular sorting.