- Do you need FACS analysis, cell sorting or both? FACS analysis results in data recorded by acquisition software. Cell sorting gives you cells separated into pure populations and all the data acquired during sorting.
- To start using analysis cytometers sign up for training by sending e-mail to Ewa.Menet@yale.edu even if you have previous flow cytometry experience. You will be required to fill out the New User Form and after completing a training session will be able to book the time on analyzers through yaleflowscheduling.com. Helpful information before going to training can be found here
- If all you need is to sort cells, determine the instrument that is most suitable for your fluorochromes of choice and contact our cell sorter operators at the links below. “Fluorochromes and filters” on Instrumentation page is a summary of our Facility’s capabilities and shows which cytometer can be used for particular fluorochrome.
- Cell Sorting is performed mostly by Facility staff. FACSAria sorters can be operated by trained users. Please contact Geoff Lyon at (203)737-5959 or (203) 737-6471 for FACSAria cell sorter training. For scheduling of your sorting time please stop by room S617 for MoFlo, FACSAria or Sony Reflection, Amistad 416 or room 345A at 300 George St. for FACSArias.
- First time sort users are required to fill the Biosafety Questionnaire.
- No primary human materials will be accepted for sorting unless documented proof (such as a test slip) of patient's negative results of testing for HIV, Hepatitis B and Hepatitis C will be provided prior to sort. This must be clear before time is reserved. Only BSL1 material (no cells of human or primate origin, no virus or retrovirus infected cells) can be brought for sorting on MoFlo.
- Sorting times cannot be booked on the internet, but can be viewed on yaleflowscheduling along with all other facility’s reserved resources. For cancellation user needs to contact cell sorter operator. Please take time to read Policies section for cancellation rules.
The following steps are in Becton Dickinson Application Note for selecting reagents for Multicolor Flow Cytometry:
- The Basics: Know your instrument. The type and number of lasers and detectors dictate whether the optical system can excite a given fluorochrome and properly detect a given combination of fluorochromes.
- Fluorochromes: Go for the bright, but minimize spillover.
PE is brighter then APC, APC> PE-Cy5> Alexa Fluor 647> PE-Cy7>PerCP-Cy5.5>Alexa Fluor 488> Pacific Blue>FITC> AmCyan> APC-Cy7> PercP>BD APC-H7
Not all fluorochromes listed above are read in the same detector.
- Colors and specificities: Define winning combinations.
The brightest fluorochrome should be assigned to the antibody binding to the protein with lowest expression. Generally, PE and PE-based energy transfer conjugates have the brightest fluorescence. In the case of cells with high autofluorescence, allophycocyanin (APC) yields more relative brightness than PEs. The combination of PerCP and PE as labels is ideal choice for measuring a low-density antigen (PE label) on a subpopulation defined by another antigen of medium or high density (PerCP label). There is no significant spectral compensation needed between PerCP and PE. PerCP conjugates provide their optimal sensitivity with an excitation laser power below 20 mW and therefore are not recommended for use in sorting experiments on MoFlo.
- On BD LSRII cytometers equipped with 532 nm lasers (LSRII Green, LSRII at Amistad, LSRII at 300 George Street), APC-Cy7 and PE-Cy7 should not be used together.
If expression is very low, it may, however, be necessary to test antibodies conjugated to a range of different fluorochromes as conjugation procedures are variable and it is not always possible to predict brightness from photochemical principle alone.
Acidic buffer conditions should be avoided during the analysis of samples stained with FITC because the fluorescence of the dye and it’s conjugates is pH dependent. Most fluorescent labels are light sensitive. To prevent artifacts, samples should be kept away from bright light.
Our facility does not perform cell preparation and staining. Users come with stained and filtered cells.
Biolegend web tools: Fluorescence Spectra Analyzer, Fluorophore Equivalency, Flow Cytometry troubleshooting etc.
Archive of BD Biosciences webinars: Multicolor flow cytometry, stem cells etc.
If you could not find the information you need, e-mail us with your questions:
|About FACS Analysis:|
|Cell Sorting: FACS Aria at LEPH or Amistad
|FACSAria at 300 George St.
|Beckman Coulter MoFlo||Gouzel.Tokmoulina@yale.edu|
|FACSAria at TAC