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How long will my sort take?

How many cells do I need?

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 samples that is to be sorted 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% OR cell culture media OR RNA/DNA extraction specific buffer OR custom buffer)

recommended with

(10-30mM HEPES – with a final concentration after sorting of cells >5mM & <30mM HEPES)

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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.