How much do the cytometers cost per hour?
The instrument price list can be viewed on the facility’s website. HERE.
How does one schedule instrument use?
Scheduling for all equipment and services is done through an online calendar. HERE.
How often are data files removed from the computers?
To keep instruments functioning efficiently, the facility staff routinely export the data files from the computer hard drives.
At the end of each day, the facility staff back up all data generated from the instruments and store them on the facility’s shared network drive. The data are then immediately deleted from the instrument’s computer.
Do you archive data?
Data stored on the shared network drive will be kept for six months. The data is kept in the form of an archive. This backup is not a substitute for saving your data in your folder on the shared network drive. Users that request retrieval of archived data will be charged. The minimum charge is half an hour, thereafter in quarter-hour increments.
Will facility staff acquire and process my data?
No. After completing the required training on a cell sorter or benchtop analyzer, the user will be able to operate the instrument independently. However, facility staff can be available by appointment for an additional session of assisted analysis.
I have used a similar flow cytometer in my previous work. Do I still need to be trained?
Yes. To maintain the quality and consistency of our service, we require all new users to be trained by facility staff and complete the required documentation before a user account is created.
What type of samples can be run on the flow cytometers?
The cytometers can reliably analyze cells, bacteria, yeast, algae, chromosomes, microorganisms such as protozoa (or malaria parasites), blood cells, and other cells which can be relatively small and in liquid suspension can be measured by flow cytometry.
How should I handle adherent and sticky cells?
Cells that are adherent and sticky must be in single-cell suspension before and during the process of acquisition. Accutase cell detachment solution can be purchase from Innovative Cell Technologies, Sigma, and eBiosciences is an excellent additive to media to keep cells or particles in suspension. The user should test Accutase on their samples before running an actual experiment. Filtering alone is not enough to keep cells from clustering or forming clumps.
Can you provide links to protocols in flow cytometry?
The Cornell University Flow Cytometry Facility provides a comprehensive list of protocols from numerous website links. SITE
Can I run any dye on the instruments?
Running any particular dye on any of the instruments will depend on the absorption and excitation spectrum of that specific dye. The facility’s cytometers are equipped with various laser lines. Please visit WEBSITE to get a full list of lasers and their corresponding filter sets. You can also check out Fluorescent SpectrumViewers from BD Biosciences, ThermoFisher Scientific, BioLegend, Fluorofinder, and others for absorption/emission information.
I have potentially infectious specimens that are considered BSL2 or BSL2+. Can I sort or analyze them?
The FACSAria, Sony MA900, and FACSMelody are enclosed in BSL2/BSL2+ biosafety cabinets allowing for infectious or biohazardous specimens to be analyzed/sorted. Every new instrument user as well as those returning with new projects are asked to fill out a registration form detailing the types of experiments planned. If the planned experiments involve BSL2/+ pathogens then necessary precautions will be taken by the facility staff per the SOP approved by Cornell EHS.
For analytical FACS on potentially infectious samples: All benchtop cytometers are housed in BSL1 rooms at the BRC and VRT facilities. Any pathogens contained within specimens for analysis MUST be inactivated before they are brought into the facility. 1 – 2% paraformaldehyde (PFA) is recommended. Dilute only the amount of PFA needed per experiment to 4% PFA from 16% stock with PBS. Store the undiluted stock at -20°C until needed (Keep stocks only for one month). Add an equal volume of the 4% stock to samples for a final concentration of 2% PFA. Finally fix cells for 15 to 30 minutes on ice, and then wash twice with PBS. Some dyes might not be compatible with fixation. For example, PE-Cy7 must be acquired within four hours of fixation.
Note: It is recommended that all fixed samples be analyzed as soon as possible. However, fixed samples can be stored for several days if needed, unlike unfixed specimens. Samples labeled with dyes such as PE-Cy7 should be analyzed within a few hours of fixation. Samples must not be left in PFA overnight as this will significantly increase autofluorescence. Always prepare fresh 2% PFA.
Why do we need to perform compensation?
When running a multi-color experiment, the emission of one fluorochrome spilling into the detector of another fluorochrome detector will impact the quality of your data. Compensation removes the spillover signal of a specific fluorochrome from all other channels.
Tips for setting up proper controls in a multi-color experiment:
- Use a single color for each compensation control.
- A control tube must contain a negative and positive cell population to compare Mean Fluorescent intensities (MFIs).
- The negative and positive populations must have the same autofluorescence background.
- Use bright markers for setting compensation.
- The compensation control samples MUST have the same fluorochrome as the experimental sample.
- Tandem dyes have lot-specific compensation. Compensation must be redone if the user switches to a different manufacturing lot.
- Compensation beads can replace cells. Negative and positive beads MUST have the same autofluorescence. Compensation (capture) beads can be used with the same fluorochrome-conjugate lot used in the actual experiment.
- For “Guides and Tips: Panel Design” please visit technical Site
How can I get help during my appointment?
Monday through Friday during normal business hours, you can call our facility phone number at (607) 255-1721 to get remote assistance during your appointment. Only experienced users should schedule weekend analyzer or evening sorter appointments
How often are the instruments cleaned and calibrated?
The sorters are calibrated daily and cleaned with bleach and water between users and before the daily shutdown. Additionally, all sorters are flushed with ethanol weekly and flushed with bleach monthly. The sorters are checked for sterility at the end of each week. Samples from the sorters are tested weekly to ensure the fluidics do not show contamination with bacterial or fungal growth.
Similarly, the analyzers are calibrated daily and cleaned with wash and/or bleach solution between users and before the daily shutdown procedure. The FACSymphony, BRC Attune and VRT Attune analyzers are flushed with bleach monthly.
How can I access FlowJo or FCSExpress?
FlowJo and FCS Express are the two common software used to analyze the .fcs files generated from flow cytometers. We have a single license of each, accessible for free for all users. Alternatively, you can inquire about purchasing your own license for you or your lab at discounted site license pricing. Please contact us via email at brc_facs@cornell.edu to get started with either free access or site license purchasing.