Frequently Asked Questions

General Questions

1. How do I sign up for a run?
   Access the FCR PPMS booking page and click on the instrument you want to use. This will open the instrument’s schedule, where you can select the amount of time you want to reserve by checking the boxes next to the free time on the calendar. Once you have selected the time you want, click the ‘Book the selected session’ button at the bottom of the schedule.
   
   For the Sorters: There are additional steps if you are reserving time on a cell sorter. After you click the button to book your session a window will pop up informing you that the reservation is pending until you fill out a sort request form (to which there is a link) and asking you to indicate which nozzle size you intend to use for your sort. Open the sort request form link and indicate your nozzle size, then click ‘Complete Booking’ at the bottom of the window.
2. How do I get after-hours access?
   For the LSRs and Fortessa: After-hours access is granted to users that have accrued a minimum of 20 hours instrument usage time in the past 12 months. If you want to know how many hours of instrument usage you have, send an email to one of the FCR staff. If you are confirmed to have 20 or more hours, fill out our After-Hours request form and you will be contacted to set up your training session. After-hours training is completed in two parts. After the first part of the training, you will need to fill out our After-Hours access practical request form. After-hours access will be yours once the practical is completed successfully! Door access may take a week to be granted to your ID.
   
   Please see the FAQ sections for the Celigo, Image Stream, and Seahorse to find out about after-hours access for those instruments.
3. How do I cancel an appointment?
   To cancel an appointment go to the instrument schedule on which your reservation is made. Click your reservation on the calendar and a window will come up with details about your reservation. Click the ‘Cancel Session’ button at the bottom of the window.
   
   If you need to cancel less than 48 hours before your sort or 24 hours before your LSR reservation you will be charged for your reserved time. You will not be charged if another user makes use of that time. **For sorts: please send an email to the FCR staff to inform them you have cancelled.**   
4. I have an emergency/last minute sample, how do I schedule time for it?
   For Sorts: Contact the FCR staff to see if there is staff availability. A staff member will contact you to schedule a time if the sort is possible.
   
   For Analysis: Schedule time on your desired machine’s calendar as soon as you need it. If you do not have after-hours access and want to run after-hours, contact the FCR staff to see if arrangements can be made, or see if a lab mate with after-hours access will accompany you (they will then be responsible for seeing that the machine is shut down properly.) No guarantees are made by the staff (i.e. no staff member is required to stay late) if you do not have after-hours access.
5. What happens if I run long?
   If the sort operator is available and the time on the instrument schedule is free you can run long. Take note that you will be charged for any time used. The operator will end the sort based on their scheduling restrictions or when the investigator is satisfied.
6. What happens if I am late?  
   You will be charged for scheduled time even if you are not present, and must yield the instrument to any user scheduled after you. If you are late for a sort, PLEASE send an email to the FCR staff!
7. What training is required in order to use the instruments?
   **All training request forms for the FCR can be accessed via our PPMS training request page**
   LSRII and Accuri (analysis): Investigators must attend the intro lecture and then complete two successive training sessions before they are able to book time and run experiments on the LSRIIs without assistance. The Accuri only requires one training session for unassisted use.  After-hours training is available after user experience requirements are met and will allow an investigator to use the machines outside of the 8:30 AM to 5 PM window.
   AriaII and S3e (sorting): Investigators who wish to have cells sorted for the first time at the FCR must attend the intro lecture and consult with a staff member before sorting. No training is required as all sorts are conducted by FCR staff.
   CyTOF (mass cytometry): All samples on the CyTOF are run by FCR staff, no user training required. Consultation with staff is required before using this machine.
   Imagestream (ISX, imaging cytometry): Investigators interested in Imagestream usage need to attend the intro lecture and then complete an introductory training session. After-hours access training is available after user experience requirements are met.
   Nanosight (microparticle analysis): Attendance of the intro lecture is recommended. Users must complete a Nanosight introductory training session.
   Celigo (imaging cytometry): Investigators who wish to use the Celigo must complete an introductory training session.
   Seahorse (metabolomics): Investigators must complete an introductory Seahorse training session. Training for this machine is typically done in groups.
   Luminex (immunoassay): Investigators who wish to make use of the Luminex must complete an introductory training session. The Luminex training request is located on the FCR’s PPMS training request page, but the Luminex is housed and managed by the RHIC.
   
   Once a training request is submitted a staff member will reach out to you shortly thereafter to schedule a time for the session.
8. How much do the cytometers cost per hour?
   Go to the FCR’s Fees and Billing page for pricing. Policies and user guidelines are at the top of the page, exact prices per hour for each machine type are at the bottom.
9. What controls do I need?  
   What controls are needed is very dependent on the experiment being run.  At the minimum you should have a negative control (unstained/no fluorescent protein) and a positive control.  You might need compensation controls depending on how many colors you have in your panel. FMO controls are worth considering. Reach out to the FCR staff if you have any questions or concerns about the controls you should have.
10. How many cells do I need to bring?
    How many cells you need to bring depends on the experiment you are running. Investigators regularly record between 10,000 and 2.5 million events or more! The rarity of your population of interest is a major driving force in determining how many cells need to be run. Aspects of the experiment being run (such as technical or biological replicates) will also influence the decision of how many cells to bring and record. Keep in mind that sample concentration and volume will affect how long it takes to analyze or sort a sample.
    
    All of these considerations apply to sorting and analysis. Sorting adds the consideration of how long it will take and how many cells an investigator needs to continue with the experiment, and often requires balancing between sample sorting time, collection tube yield, and total sort time booked. It is recommended this detail be worked out with a PI or lab mate involved in the experiment.
11. How long will it take to sort my cells?  
    This depends on the nozzle size being used for the sort. Our most commonly used nozzle, the 85um, sorts at a rate of ~1mL per hour at an appropriate concentration. Full information on sort time and nozzle size is available on our Sorting page. In general, larger nozzle sizes take longer to sort and smaller nozzle sizes take less time to sort. Different nozzle sizes have different maximum sample concentrations!! 
12. What is a GNT number?
    This is a number that the IBC assigns to a project when it will involve the use of recombinant DNA or BSL 2 (or higher) precautions. GNT numbers are formatted like so: Bushnell-15-028, the first part being the PI’s last name, the second part being the year the number was assigned, and the third part being an indication of the sequence in which that number was assigned. GNT numbers must be provided on sort request forms for these types of experiments so that the FCR can verify with the IBC that samples from the experiment can be sorted without risk to personnel or without additional safety precautions being taken. The FCR will refuse to conduct a sort if the GNT number cannot be verified before the scheduled start time.
13. What do I need to do differently to sign up for a BSL 2 sort?
    You will need to provide a GNT number on your sort request form. The request form and instrument booking will not be approved until you provide a valid GNT number for verification. If you do not know what the GNT number for your project is, your PI should know. Refer to the FAQ ‘What is a GNT number’ for further information.
14. Can I request a specific operator for my sort/assisted run?
    Specific operators will not be assigned to sorts or assisted runs based on user preference. If you want a specific operator you can request them but there is no guarantee they will be available to do the sort or run.
15. Will the FCR analyze data for me?
    The FCR will analyze data for a fee (we encourage you to try it for yourself!) Contact Matt Cochran if you would like to discuss having the core analyze data for you.
16. When should I filter my samples? 
    Samples should be filtered directly before coming to the FCR to sort or right before putting the sample on the instrument.
17. What should I do if another user will not vacate a machine during my reserved time?
    Immediately find a staff member to help resolve the situation. 
18. What should I do if I am not happy with the quality of my sort?
    Contact the operator who conducted your sort and Matt Cochran to start a discussion and set up a time to review your experiment on the sorter.
19. Will the FCR help me design a panel for analysis or sorting?
    The FCR will happily consult with you on panel design! Contact Matt Cochran or the FCR staff to discuss your needs and set up a meeting.
20. Where can I find information on training/the intro lecture?
    Go to our Policies and Procedures page and scroll down to the ‘Access and Training’ section. 
21. What should I do if I don’t think the machine I am using is working correctly?
    If you are an experienced user, you are welcome to try some of the basic troubleshooting gone over in the instrument training. If you cannot get the machine to work or if you are a new and inexperienced user, please post a message into the Slack messenger right away! We prefer if you ask us for help sooner than later so your instrument time is not wasted. When in doubt, send us a message and we will be more than happy to help.
22. How do I tell where my cells are on a FSC vs. SSC plot?
    This can be quite tricky, especially if you have not worked with your cells before. First try to get everything on scale if you can, then adjust each voltage higher until you can start to see your populations (typically areas on the plot where events are more densely concentrated.) If you can start to see your populations, great—make some fine adjustments to your FSC and SSC voltages if you want, and continue gating. If you still having trouble finding your cells, find a staff member to help you before too long. There are a few simple strategies for verifying if what you have gated on your FSC vs. SSC plot are your cells or not.
23. How do I know if a detector voltage is too high or too low?
    Put simply: if your signal is on scale, your voltage is not too high or too low. The reality of this question is that not only is the decision of where to set a detector voltage a bit subjective in the end, but it can be influenced by the other fluors you are working with in your panel, the brightness of your fluor in question, and the abundance of the cell marker you are trying to bind. This approaches more advanced aspects of cytometry and investigators are encouraged to contact the FCR staff to discuss and find out more!
24. How do I know if an LSR/Fortessa is clean?
    There will always be a little bit of background when collecting data on a flow cytometer. On a correctly functioning instrument the scatter plot should show relatively few events positioned mostly near the origin. Some of the background events may come out on the FSC or SSC scale a little bit, but as long as the events are not too abundant the system should be OK. When acquiring a clean tube of water, the events per second readout should be somewhere between 0 and 45 (this will vary based on the machine, the type of water used, etc.) The second thing to check to ensure cytometer cleanliness is that there is no significant fluorescent signal being picked up by any of the detectors. If the machine seems dirty, either because of high background or clear carryover from a previous sample, try priming the instrument and running a clean cycle on it (2 minutes each of bleach, ethanol, and water in that order.) If that fails to get the system looking better, find a staff member to help you out.
25. When should I prime the LSR/Fortessa?
    Priming helps to eliminate bubbles in the flow cell, minor clogs, and leftover debris/carryover from samples. Most users prime the machine when they sit down to use it, which is recommended. If you lose signal in the middle of running a sample, taking the sample off and priming is always a good first option. Run a bit of water after you prime the machine, then check your sample again.
26. Can I use a machine after-hours if I am with someone who has after-hours access?
    Investigators who do not have after-hours access are allowed to run after-hours if and only if they are accompanied by a user with after-hours access that has agreed to assist. The user with after-hours access should be the one to reserve time on the machine, and at the least should be present for the entire run to ensure the machine is started and shut down properly. Responsibility for appropriate care of the machine falls on the user with after-hours access. It is not permitted for users with after-hours access to simply give their IDs to users without it so they can access the FCR. Consequences will occur if this protocol is not followed. Visit our Policies and Procedures page for more information.
27. Do I have to stay in the FCR for the entire duration of my sort?
    You are not required to stay at the sorter with the operator for the full duration of the sort, nor the FCR for that matter. As long as sufficient contact information and instruction on when to contact are left with the sort operator you are free to leave and tend to other business.
28. What is a post sort?
    A post sort is an assessment of collection tube purity and general sort quality/effectiveness that is performed after completion of the sort upon investigator request (there is a field in the sort request form for this.) The machine is cleaned and a collection tube of each sorted population is run on the cytometer and a specified number of events are recorded (default 2,000) and displayed on the plots used for the sort to see where the sorted populations fall within the gates. This can reveal (for example) if your cells are dying excessively after sorting, if there are contaminating cells being sorted, and if cell viability is holding up after the sort (ideally the post sort will reveal a pure fraction of viable cells!) Post sorts can help identify and address sort issues. Post sorts cannot be performed out of plates, slides, or lysis buffer. Information on post sorts can also be found on our Sorting page.
29. What is a sort mask/precision mode?
    Precision modes (or sort masks) are different sets of priorities that the sorters will use when making sort decisions. Purity, Yield, and 4-way Purity are the three commonly used precision modes on the Arias (purity and yield are paralleled by purity and enrich on the S3e.) Purity will give you higher purity collection tube at the expense of a few cells of interest if sorting them would also cause undesired cells to be sorted with them. Yield is the converse of that, sorting as many cells of interest by potentially sorting some undesired cells. More detailed information can be found on the Sorting page.
30. Does the speed with which I run my sample affect my data?
    It certainly does. Running your samples at faster speeds will has the potential to increase the spread of your data. When a sample is run at a high speed the events going through the machine have a greater likelihood of not passing directly through the lasers of the machine, thus potentially giving you a measurement that is skewed slightly higher or lower on the detector scale. This can affect both scatter and fluorescence data. Running samples as slow as you can (ideally on the lowest flow rate the cytometer is capable of) will minimize the spread of your data due to the phenomenon just described. Sample concentration is a determining factor in how fast a sample can be run. Contact the staff for more information.
31. What should I do if the software is not working correctly?
    Users typically encounter the software freezing or not allowing them to edit the names of elements (such as tubes, specimens, detector labels, etc.) Oftentimes restarting the software will fix whatever general issue you are experiencing. Software that users encounter in the FCR (FACSDiva, Imagestream, celigo, nanosight) does a good job of keeping your data if you need to restart in the middle of a run unless there is some catastrophic failure (a rare occurrence.) You may need to open the task manager by pressing ctrl+alt+delete and close the software that way if it is really misbehaving. FACSDiva has a bad habit of snapping to only one of two monitors—if this happens to you just click the edge of the window and drag it back to full size. If you are unsure or if the issue is more serious do not hesitate to grab a staff member to help you out.
32. How will my reservation be billed? Is there a minimum Charge?
    Reservations are billed by time used or time booked, whichever ended up being greater (i.e. if you finish early you are billed for the whole reservation, and if you run long you are billed by the minute for the entire time you were logged into PPMS on that instrument.) There is not a specified minimum charge, but there is a minimum amount of time you can book on the instrument calendars, which is 30 minutes for the analytical machines and 1 hour for the sorters. See our Fees and Billing page for further information.
33. What changes when different nozzle sizes are used on the Aria?
    When you change nozzle sizes on the Aria, the obvious thing is that the size of the nozzle, and therefore the maximum size of cell you can fit through that nozzle, changes. The operating pressure of the fluidics also changes, higher pressure for smaller sized nozzles, lower pressure for larger sized nozzles. 
34. How do I get my data?
    Data generated on FCR instruments is placed onto the university’s data transfer pipeline, which has a folder for each PI, listed last name first. On the analyzers, the users themselves will transfer their data (this process is explained during training.) On the sorters the operator is the one who performs the transfer of all data generated during the sort.
35. How do I access the transfer pipeline?
    Your PI’s transfer pipeline folder should be accessible from any computer in your lab. Your PI should be able to show you how to access this folder. If you have a computer running Windows, open the Run app and paste this address in: \\smdnas\FCC_Transfer
    (or click the link if you are on the URMC network!!) Find your PI’s folder, open it, and grab your data. If you are having trouble accessing or transferring your data for any reason, please contact the FCR staff. **Note: Those in labs on river campus will have to use Box, dropbox, or some other form of cloud storage to transfer their data, as the firewall between river campus and the medical center does not allow access to the transfer pipeline. Flash drives are not allowed due to the potential transmission of malicious software.**
36. Is my data backed-up?
    It is! The FCR goes to great lengths to ensure the maintenance and integrity of user data. The FCR keeps extensive archives of all data generated on instruments under its purview. If you need data from the FCR archives, contact Matt Cochran. There is a $20 fee associated with retrieval of data from the archives. Data is kept on the instruments’ local machines for a week on the analyzers and for a month on the Sorters—if you do not transfer your data in that timeframe, you will need to have it pulled from the archive.

ImageStream specific questions

37. What are the capabilities of the system?
    1. The ISX is a 12-channel instrument. Two channels are used for Bright Field images and 10 can potentially be used for fluorescent markers, if not collecting dark-field (side scatter-like) images.
    2. There are three magnification options: 20x, 40x, and 60x. The resolutions are approximately 1um/pixel (20x), 0.5um/pixel (40x), and 0.3um/pixel.
    3. There is a 96-well plate loader option for unassisted runs (finicky!)
    4. The maximum acquisition speed is theoretically about 1,000 events/sec, however optimal speed (depends on the size of the objects) is between 200-600 events/sec.
    5. The minimum required sample volume is at least 50ul, 65ul is recommended. The volume taken from the sample is less than 30ul. To collect the entire volume of the sample will take about 20-22 minutes. IT IS NOT NECESSERY TO COLLECT ENTIRE VOLUME OF THE SAMPLE!!!
    6. The optimum cell number per sample is between 1-3 million, so about 20-60 million per mL. Less is fine, but it will take longer to collect a sufficient number of cells per sample. 10,000 images per sample is usually considered sufficient.
38. How do I sign up for a training?

To sign-up for ISX training please use PPMS.

39. May I bring my own samples to the training?

Yes, that is recommended but not necessary. Please contact Wojtek to discuss details (via email.)

40. What should I bring to the ISX training?

Nothing is required if you are not bringing your own samples.

41. How long does it take to get trained on the ISX?

The required training will take up to 2 hours if you are bringing your own samples, or about 30 minutes without them. The optional follow-up training sessions are as needed, assisting with run setups, intro to data analysis etc.

42. How do I transfer ISX data?

The ISX instrument computer is not on the URMC network, so the data needs to be transferred using the analysis computer located in the same room. PI’s transfer folders (on the transfer pipeline) are used to move files. 

43. How do I analyze ISX results?

ImageStreeam data is analyzed using Amnis/Luminex IDEAS software. The introductory training and data analysis support is provided by the FCR.

44. Is IDEAS software free?

Yes and no... The “old” free version 6.2 is provided by the FCR. The version capable of machine learning based analysis can be purchased from Amnis/Luminex. Access to this version for FCR users is being evaluated(???)

45. Can I install IDEAS on a Mac computer?

There is only a Microsoft Windows-based version of IDEAS. To run it on a Mac it is necessary to either set up double boot with Windows environment or use Parallels or a similar type of software. The FCR does not support those solutions.

46. Can I have after-hours access to use ISX?

Yes. It requires a minimum of four hours of independent experience on the instrument and a short training. Please sign up for after-hours training using PPMS.