Flow Cytometry Core
Frequently Asked Questions
- Can I sort unfixed human samples?
Yes. A BioProtect III Walk In Clean Air and Containment Cabinet houses the FACSAria II. The BioProtect III is a specialty type Class II biological safety enclosure designed to offer personnel, product, and environmental protection from potentially aerosolized biological hazards.
When a sort has a biohazard potential, a biohazard form must be completed prior to each sort. Both requestor and PI's signature are required. Unit 3.6, Standard Safety Practices for Sorting of Unfixed Cells, of the Current Protocols in Cytometry, is available at \\LRI-fs01\RESEARCH\FLOWCYTOMETRY\SORTING_BIOSAFETY\Scan001.pdf.
- Can I run fluorochrome X with fluorochrome Y?
To determine the spectral overlap of varied fluorochromes visit www.bdbiosciences.com/spectra/ in the Links page. Select fluorochromes from the list and compare their excitation and emission wavelengths to get an idea of compatibility. Fluorochromes whose emission peaks overlap completely cannot be used together (GFP and FITC for example).
- How many cells do I need for each tube? What should the volume be?
Analytical experiments, under ideal circumstances, one million cells per tube are standard. In many situations, few cells may be available for each of the controls and experimental samples. The minimum number of cells required is going to vary based on needs. For very simple experiments, tens of thousands of cells may be needed. Multicolor experiments that subset out small percentages of cells may require several million cells per tube in order to acquire appropriate numbers of the target populations. (ref: Cytometry A. 2008 May;73(5):384-5. Roederer, M. How many events is enough?) A minimum of 200ul volume in each sample tube is required.
- What controls do I need for my experiment?
Instrument controls consist of samples needed to compensate the different fluorochromes and to choose starting baseline voltages if optimal PMT voltages are not used. To properly compensate for an experiment, an unstained control (cells with NO fluorochromes) and a single color control for each fluorochrome for that experiment are needed. The instrument is set up using these controls for compensation. These controls are mandatory and needed every time. The individual user determines experimental controls, as in a positive and negative control, stimulated vs. unstimulated, N-1, etc.
- Do I really need all these controls?
Yes. At times, the Flow Cores is called upon to set-up the instruments without these controls. In these circumstances it is the Core?s policy to inform the investigator that the instrument is being set so the data looks a certain way based on expectations and experience. Results from these experiments are invalid and need to be repeated with the proper controls. Any conclusions drawn under these circumstances are highly suspect at best.
- Do I really need to run compensation controls everyday?
Yes. Even though the instruments are QC'd on a daily basis to measure stability and to provide researchers with platforms for reproducible data, there are always small differences in the instruments and more so in experimental set-up each day. Under proper set-up, small changes in instrument settings are noticed from day to day. When differences in your control samples compared with experimental samples are seen, it is important to be sure it's really due to design, and not due to some errant fluctuation or improper set-up.
- How long will it take to sort my cells on the Aria II?
The answer to this question will vary greatly. For the typical low-pressure sort with the 100 um nozzle, running a threshold rate of about 3000 cells/second can be estimated. This translates to about 10 million cells in one hour. For the typical high-pressure sort with the 70 um nozzle around 20,000 cells/second, translates to around 70 million cells in one hour. These estimates are conservative and speculative. Adjustments to the rate of cells/second are made based on the efficiency and quality of the sort in relation to the needs of the investigator.
- Which pressure/nozzle settings are right for my sort?,
The nozzle size depends on the size and characteristics of the cells. The nozzle size should be about four to five times that of the cells that are being sorted. For sorts targeting most lymphocytes, the 70um nozzle is used. For larger cells, the 100um nozzle is used. Increased pressure and the smaller orifice of the 70um nozzle exposes the cells to a slightly harsher environment, albeit briefly. Delicate cells require the 100um nozzle regardless of their size.