Common Recommendations
Shelley's Buffer – HBSS:
- HBSS without phenol red, Ca2+ or Mg2+
- 10mM HEPES buffer (be careful to use the cell culture variety and not just a chemical buffer)
- DNAse1 (50ug/ml) from Roche (added the same day as the sort, to minimize aggregation due to cell death)
- 1mM MgCl2
- BSA Fraction V (2.5mg/ml)
- Be sure to check your pH (BSA is acidic)
Useful Links:
Keep In Mind:
- You should match bright flourophores with rare targets, and vice versa. FluoroFinder is a good resource for panel building.
- Strain your sample as close to the sort as possible, to minimize the chance of clogging the machine. We have Biosafety Cabinets available in our labs if you are able to strain immediately before loading your sample.
- Unstained controls are important for setting up the instrument. Please bring them if possible.
- If you are viewing live cells, add a Live/Dead / Viability dye.
- Bring extra buffer to add to your collection tubes.
- Bring extra collection tubes.
- Bring any supplies you must have, especially if you require a sterile environment to work with your sample.
- An ideal concentration is 10-30 million cells per mL
- It is better to have a sample that is too concentrated than too dilute, as you can always further dilute your sample.
- Always have your downstream in mind. For example, if you are sorting for 10X analysis, certain dyes may be off limits.
- If you know what to expect from your sample, drawing a simple graph is helpful for the operator to reference.
- If you use an enzyme, adding an inhibitor is recommended. Otherwise, the enzyme may continue to effect your sample, even with after being washed.