Getting Started with Flow Cytometry

We suggest you to start by watching these fluorescence tutorials and make sure you understand the principle of the flow cytometry assay you would like to run.  Feel free to explore our Tools page where you will find useful resources. If you are not sure what assay you need to run or if you have general questions about your experiment please request a consultation by filling out this form or sending an email to flowcytometry@chop.edu. If your project will involve cell sorting please see our guidelines for planning a cell sorting experiment. We welcome walk-ins if you would like to discuss your project with us; if none of the staff is available right away we will schedule a discussion.

If you have minimal or no previous flow cytometry experience and would like to run simple experiments that require the detection of up to 4 colors we suggest using the Accuri C6 cytometer because it is easy to learn. However, there are fluorochromes that cannot be detected on Accuri. In this case, the next choice would be CytoFlex, which is still a user-friendly cytometer that can detect a larger variety of fluorescent dyes. Researchers already proficient with the LSR cytometers (LSRFortessa, LSR II) or with FACSCalibur may want to keep using these instruments since they are very reliable.

Fill out a training request form or send an email to flowcytometry@chop.edu to let us know which instrument you would like to learn to use and the date and time that would work for you.

Yes, please fill out the training or consultation request form or email flowcytometry@email.chop.edu with a brief description of your project.

Full-service and semi-assisted cell sorting may be scheduled only during business hours, which are Monday-Friday 9 a.m. to 5 p.m. Sorters may be reserved outside business hours by trained users who do not need help from the staff. Analyzers may be used 24/7 by users with a CHOP ID; outside users are not permitted to enter the building when the staff is not present. All users who need assistance must schedule their work in the flow lab during business hours.

It typically takes two sessions of 2 hours each to learn the basics of cell sorting on a specific instrument. If you sign up for training please do not plan to bring any cells for the first two sessions; we will use beads. During a third session you should schedule a real cell sorting experiment and sort your cells with assistance from a member of the Flow lab. Achieving proficiency and the ability to troubleshoot common incidents that may happen during sorting (droplet instability, clogging) will require more practice. Users who rarely need to sort are strongly advised to request full-service sorting.

Before reserving a sorter make sure the instrument you chose can detect all the fluorescent dyes you will be using. Also keep in mind that MoFlo Astrios can be used in high-speed or normal speed modes and it can sort up to six cell population in one pass. FACSJazz can be set up only with 100um nozzle (normal speed mode only), it has only six color detectors, and it can sort only two populations at a time. Both sorters can deposit cells in multiwell plates. Send a brief description of your experiment to flowcytometry@email.chop.edu or ask directly any member of the flow lab if you are not sure which sorter to reserve.

Yes, all our sorters can deposit cells in 96-well plates. Typically a plate is filled with single cells in a few minutes but if the target population to be sorted is very rare it will take longer. The sorters can be set to deposit any given number of cells in each well.

We suggest you start with a pilot experiment to check the feasibility of your project and how much time a full experiment will take. Note that Astrios set on normal speed (100um nozzle) and FACSJazz one can process 25 million cells/hour. Astrios set up with a 70um nozzle (high-speed mode) can run 100 million cells/ hour. If you know the approximate number of cells you will bring it is easy to calculate the time needed to sort. Keep in mind that additional time is needed to set up the instrument at the beginning of your experiment and for compensation. Checking the purity of sorted cells will also take some additional time.

When you request full-service you will need to bring your samples to the Flow lab and a staff member will operate the sorter. You will have to confirm at the beginning of your experiment that the gates are properly set and the target populations to be sorted are correctly identified by the operator. You may remain in the lab for the duration of the sort or you may leave if you wish. We require you to pick up the sorted cells and the collection tubes after the sort is completed.

For semi-assisted sorting the staff will set up the sorter for you but you are required to be present in the Flow lab for the duration of your experiment, load the samples and the collection tubes on the instrument, and monitor your sort. The staff will be available for the duration of your semi-assisted sort to troubleshoot any incidents.

Please note that full-service and semi-assisted sorting are available only during business hours.

For unassisted sorting users are required to perform all, including starting up and shutting down the sorter, without help from the staff. Only trained end-users may request semi-assisted or unassisted sorting. The Flow Lab reserves the right to revoke the access to semi-assisted or unassisted cell sorting to users who fail to follow the proper procedures while operating the instruments.

ShareFile is the system used at CHOP for transferring files in a secure manner to any computer connected to Internet. If you have a CHOP ID please sign up for a ShareFile account by logging on a computer connected to the CHOP network and filling out this form and check the request "ShareFile Access" box.

Researchers from Penn or other institutions may use other online storage options (e.g. Dropbox, Google Drive) for transferring files.

FACSCalibur users may use a memory stick or external hard drive to transfer the data they generate in our lab.

Cell sorting and analysis services performed by staff include temporary online storage (one year) using the ShareFile system, using the ShareFile space of the Flow lab. End users do not necessarily need a ShareFile account to download the files uploaded by the staff.

Note: The “FileShare” system (a.k.a. SAN drives) refers to the network drives that typically can be accessed only from CHOP computers. The computers connected to equipment in the flow lab are not on the CHOP network and they cannot access the FileShare/SAN drives directly. Please do not plan on using FileShare for transferring your data from the Flow lab.

In order to avoid a full charge, all cell sorting reservations during peak time must be cancelled at least 48 hours before the scheduled sort time. Reservations during peak time for all analyzers must be cancelled 24 hours before the start time of your appointment. Cancellations of off-peak reservations on cytometers and instruments will incur a charge equivalent to 20% off the reserved time.

If you miss your appointment because you are sick please bring a doctor’s note and we will void the cancellation fee for your appointment.

Please be aware that CHOP Security does not allow users to carry samples on passenger elevators unless they are triple packed (e.g. samples should be inside capped tubes, within a leak-proof bag with absorbent material, inside a closed box). CHOP employees may use the service elevator for bringing double-packed samples (e.g. samples inside capped tubes placed in a leak-proof box or bag with absorbent material) to the Flow lab.

The FACSJazz has the standard 100um nozzle permanently installed. The BioSorter can be set up with 250um or 1,000um nozzles. The MoFlo Astrios is typically set up with the 70um or 100um nozzle and for special projects the staff can set up Astrios with 120 or 150um nozzles.

As a rule of thumb, the diameter of the cells to be sorted should be at least five-fold smaller than the diameter of the nozzle. This rule will ensure that the cells will pass through the nozzle without being damaged and they will not disturb the droplet formation.

• For the 70um nozzle (high speed sorting), cells must have a diameter smaller than 14um.
• For the 100um nozzle (standard size and regular speed sorting), cells must have a diameter smaller than 20um.
• For the 150um nozzle cells must have a diameter smaller than 30um.

For larger cells and small organisms you should consider using the Biosorter.