Koch Institute

http://ki/mit.edu/sbc/flowcytometry

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FAQ

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Is the KI Flow Facility at MIT open to investigators outside of MIT?
We have opened up the KI Flow Cytometry Facility at MIT to biotechnology companies if the proposed use does not negatively affect our MIT investigators.  If you are interested in using our cytometers, please contact the facility manager Glenn Paradis at gap dot mit dot edu and he can expand upon the details of how access may be granted.

I have a lot of previous experience running the same analyzers. Do I still need to attend orientation and schedule a hands on training?
Yes, you still need to go through the system we have set up to grant you access to our cytometers.  We have over 150 active facility users that rely on our equipment each month and we cannot have mistakes made because of a lack of training that affect their work.  In addition you still need to know how to get laser detector information about our cytometers, how to book them properly and how our facility and billing policies are set up.

How do I properly prepare my samples including the correct tube, cell concentration, minimum cell volume, ect.?
The answer to this question will depend on whether you are using our analyzers or sorters.  For analyzers, see the Analyzer Training Guidelines and for sorters see the Cell Sorter Guidelines from the Forms section of our web site.  http://web.mit.edu/flowcytometry/www/forms.html

What is the fewest number of cells I need to start with?
This question comes up routinely and the simple answer is that you need enough cells to set up the cytometer properly and to collect data files.  Most labeling protocols suggest starting with 1-2 million cells.  After the required wash steps, we routinely find a 20-50% loss of cells.  For cell cycle using alcohol fixation, the loss can be 90%.  The less you start with, the more you lose along the way. If you expect to collect a 10,000 event data file (1% error), then start with at least 100,000 cells and hope for the best. If we do not have enough cells, you jeopardize the experiment so that no results will be obtained.

Do I need to bring my controls for every experiment?
Yes, the proper controls are required for every experiment.  Compensation must be adjusted for each experiment especially if PMT voltages change from day to day.  Even if PMT voltages stay the same, tandom fluorophores are know to change their fluorescence profile over time and the only way to adjust compensation is with that days controls.

How do I cancel an appointment and is there a penalty?
Email the lab staff at flowcytometry-www at mit dot edu  then if you made the appointment, delete it from the cytometer agenda.  If we made the appointment, we will delete it for you.  If the email time stamp is greater then 24 hours from the start of your appointment, there will not be a charge.  If it is less then 24 hours, you will be charged for the entire appointment as per our facility policy unless we find another investigator to take the time.

What lasers and detectors are on your cytometers?
Click on our instrumentation link and then the equipment link and you will download an Excel file with all of our lasers and detectors for all of our cytometers.

Can I run flurophore X?
Google, BD Spectral viewer or Invitrogen Spectral viewer or get the absorption and emission spectra of your fluorophore from the manufacturer.  Look up our cytometer laser and detector configurations (see answer above) to see if any of our laser lines can excite and any of our filters can detect your fluorophore.  Excitations of 50% or more are usually required to see signal unless the amount of fluorophore/ cell is huge.

Do you have computers for off line data analysis, what is the cost and how do I sign up to use them?
We do have a few data analysis computers to use for free.  FACS Diva, Cell Quest, FlowJo and Modfit are our programs used to analyze flow cytometry data.  There is no sign up for them.  There are used on a first come, first served basis.

Can I have a copy of your data analysis software to load on my PC?
We do not have a licensing system for data analysis software on remote PC’s.  You will have to purchase your own software.  Flow cytometery data analysis software is a great way to spend the last few dollars in your grant.

Can I run yeast or bacteria on your analyzers and sorters?
Yes, we do run yeast and bacteria on our analyzers and sorters.  A rigorous cleaning process is implemented between users to avoid contaminating the next users samples.  Any time spent doing the extra cleaning is billed to the user with the bacteria or yeast.

Do I need to filter my cells before I run them?
It is required to filter any sample on a sorter just prior to loading it.  Expensive damage to the sorter can occur when it clogs and deflection plates arc ($10K+ to replace a high voltage board).  A $1-2 filter does not seem like a large price to pay compared to the expensive repair and down time of the sorter.

Analyzer filtering can be important as well. If you are trying to avoid the cost, you can run the sample unfiltered, if after vortexing, you do not see cell clumps.  If after loading a sample, the cells stop running, you must filter the sample to avoid clogging the sample injection tube (SIT).  A clogged SIT will ruin the next person’s appointment if the lab staff have left for the day.

How many cells can be sorted in 1hr?
The sorter takes about 15-30 minutes to set up for your sample.  Once the sort starts, we will typically run at rates of about 15K-20k events/sec.  If the sorter clogs, it can easily take 30 minutes to unclog and recheck the laser alignment. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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