Frequently Asked Questions
Please feel free to either scroll down to view all the questons or click on the link for each question under Frequently Asked Questions.
Can I run fluorochrome X with fluorochrome Y?
To determine the spectral overlaps of varied fluorochromes please visit the Spectrum Viewer pages in the Helpful Links page. There you will be able to pick fluorochromes from a list and compare their excitation and emission wavelengths to get an idea if they are compatable. Fluorochromes whose emission peaks completely overlap can't be used together on our instruments, GFP and FITC for example. If you are still stumped, feel free to contact us regarding your choices.
How many cells should I put in each tube for my experiment? What should the volume be?
Under ideal circumstances one million cells per tube for analytical experiments is standard. Under many situations you may not have the luxury of that many cells for each of your controls and experimental samples. The minimum number of cells required is going to vary based on your needs, for very simple experiments you many only need tens of thousands of cells. 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. You'll want to have a minimum of 300 ul volume in your samples.
What controls do I need for my experiment?
When you think controls, you need to separate your thoughts into Set-up Controls and Experimental Controls. Set-up Controls consist of samples needed to balance out the different fluorochromes and to choose starting baseline voltages if you are not using optimal PMT voltages. To properly compensate for your experiment you will need to bring an unstained Control (your cells with NO fluorochromes) which we may not include in your set-up but will want to see, and then a Single Color Control for each fluorochrome that you are using in your experiment. The instrument is set up using these controls for compensation; without them we would be guessing at these critical settings. These controls are manditory and you will need them every time you set up your experiment. Experimental Controls would be determined by the individual user, as in a Positive and Negative control, Stimulated vs. Unstimulated, etc. We can help you with these experimental design questions if you need help.
Do I really need all these controls?
Yes. We get called on to set-up the instruments without these controls at times. In these circumstances it is our policy to inform the investigator that we are no longer doing science, we are setting the instrument so the data looks a certain way based on our expectations and experience. Results from these experiments are invalid and need to be repeated with the proper controls, any conclusions drawn under these circumtances are highly suspect at best. If you are going to spend the time and considerable money required for flow cytometry experiments please take these steps to make your data meaningful.
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 insure we can provide researchers with platforms for reproducible data, there are always small differences in the instruments and more so in your experimental set-up each day. Under proper set-up you will notice small changes in instrument settings from day to day, so small that you might conclude that Set-up Controls aren't really that important everyday. This is faulty logic, when you see differences in your Control Sample compared with your Experimental Samples, you'll want to be sure it's really due to your design, and not due to some errant fluctuation or improper set-up.
How long will it take to sort all my cells on your sorter?
The answer to this question will vary greatly. You can make some rough estimates that should put you in the ballpark. For the typical low pressure sort with the 100 um nozzle you can estimate that we'll run a threshold rate of about 3000 cells/second, this translates to about 10 million cells in one hour. For the typical high pressure sort with the 70 um nozzle you can estimate around 20,000 cells/second, translating to around 70 million cells in one hour. These estimates are conservative and speculative, we'll make adjustments to the rate of cells/second based on the efficiency and quality of the sort in relation to each investigators needs and the situation.
Which pressure/nozzle settings are right for my sort?
We will choose the nozzle size for your particular sort. This will depend on the size and characteristics of the cells more than anything else. The nozzle size should be about four to five times that of the cells that are being sorted. For sorts targeting most lymphocytes we'll probably run the 70 um nozzle. For larger cells we'll probably choose the 100 um nozzle. Increased pressure and the smaller orifice of the 70 um nozzle will expose your cells to a slightly harsher evironment, albeit very briefly. If you cells are more delicate, we might choose the 100 um nozzle regardless of their size.
Can you find my data from 3 years ago? No, I don't know what the experiment name was...
We hope so. Please realize we're not in a position to know where every bit of data is prior to September of 2006. Bring all of the information you have regarding older experiments to help us try to track them down, and please be understanding that we might not be able to drop everything we are doing and find your data that instant. We will do everything we can to find your data.
What is your policy for billing times that are slightly over or under the amount of time that was originally signed up for?
We are very understanding and forgiving for times that go over and under. You will be billed for the amount of time you sign for unless you go significantly over your time and if you use much less than you signed up for we *may* decrease your time. Five to 10 minutes over your original time is not a big deal unless you have someone scheduled immediately following you(which is usually the case). This is based on a case by case basis but we'll be understanding for occasional issues that come up regarding this.