Siteman Flow Cytometry Core (SFC)

This cornerstone of the core allows for four detectors off a 200mw 488nm blue laser, up to three detectors off a 100mw 640nm red laser, and up to three detectors off a 100mw 405nm violet laser. A krypton-ion laser is also available for configurations involving 350nm (UV), 530nm (green), 568nm (yellow) and 752nm (NIR). Adequate notice is required for optics changes on the krypton laser as well as up to an hour allotted for the changeover time. A Propel Labs Co-Lase tower allows simultaneous use of four lasers and eleven emission detectors. The MoFlo is best known for the lowest cell sorting shear factor and small sample accuracy, as it has a manual sample station for minimal sample dead volume.

The Sony SY3200 “Synergy” cell sorter is known for its very accurate high-speed sorting, pushing the Poisson limits for speed while maintaining excellent viability and purity results. In addition, unique reflective collection optics with over 375um laser intercept spacing and the newest “sputtered” hard coating emission filter technology have combined to attain new benchmarks for sensitivity and laser isolation. This instrument has two HAPS (highly automated parallel sorters), which can perform two different experiments simultaneously. Each HAPS module has 21 colors and five laser wavelengths: 355nm (UV), 405nm (violet), 488nm (blue), 561nm (yellow) and 640nm (red). The sorter modules are housed in a Class II biological safety cabinet, which has a unique passive aerosol evacuation feature that is as robust as the Baker hood itself. Also, each HAPS module is capable of four-way sorting and plate deposition.

The Aria SORP has choices of 70um, 85um, 100um, and nozzles, and is equipped with an ACDU plate robot.  Laser wavelengths are 355nm, 405nm, 488nm, 561nm, and 640nm, and the sorter has 18 fluorescent detectors.

The Thermo Fisher Bigfoot Cell Sorter features up to 7 lasers, including UV (355 nm), violet (405 nm), blue (488 nm), green (561 nm), yellow-green (592 nm), red (637 nm), and near-infrared (808 nm), enabling detection across a wide spectral range for advanced multi-parametric analysis. It supports high-speed sorting at up to 100,000 events per second with sterile, multi-well plate sorting capabilities.

5 Laser full spectrum sorter 16UV-16V-14B-10YG-8R Coarse Wavelength Division Multiplexing (CWDM) semiconductor detector arrays for efficient spectrum capture of dyes emitting in the 365-829 nm range. High bandwidth electronics design up to 67 channels. Supports 6-way sorting and multi-well plate sorting.

• Blue laser (488nm excitation) FL1 (530nm emission centering wavelength/30nm bandwidth)- Alexa 488, FITC, GFP, YFP, CFSE
• Blue FL2 (585/42)- PE, Propidium Iodide
• Blue FL3 (620/20)- PE/Texas Red, ECD
• Blue FL4 (695/40)- PerCP/Cy5.5, 7-AAD
• Blue FL5 (740 LP)- PE/Cy7
• Red laser (637nm) FL1 (660/20)- APC, Alexa 647
• Red FL2 (725/20)- Alexa 700
• Red FL3 (740 LP)- APC/Cy7, e780
• Violet laser (407nm) FL1 (450/50)- Pacific Blue, Brilliant Violet 421
• Violet FL2 (545/30)- Pacific Orange, Krome Orange, Live/Dead Yellow

• Blue laser (488nm excitation) BL1 (530nm emission centering wavelength/30nm bandwidth)- Alexa 488, FITC, GFP, YFP, CFSE, Live/Dead Green, DyeCycle Green, CellROX Green
• BL2 (695/40)- PerCP/Cy5.5, PE/Cy5.5, PE/AF700, PerCP, 7-AAD
• Red laser (637nm) FL1 (670/14)- APC, Alexa 647, SYTOX Red, FxCycle Far Red
• RL2 (720/30)- Alexa 680, Alexa 700
• RL3 (780/60)- APC/Cy7, e780, Live/Dead Near – IR, DyeCycle Ruby
• Violet laser (405nm) VL1 (450/40)- Super Bright 436, Brilliant Violet 42, eFluor 450, Pacific Blue, BD V450, VioBlue
• VL2 (525/50)- eFluor 506, Brilliant Violet 510, Pacific Green, BD HorizonV500, VioGreen
• VL3 (610/20)- Super Bright 600, Brilliant Violet 605, Pacific Orange
• VL4 (710/50)- Super Bright 645, Brilliant Violet 650
• VL5 (660/20)- Super Bright 702, Brilliant Violet 711
• VL6 (780/60)- Brilliant Violet 786
• Yellow Laser (561nm) YL1 (585/16)- PE, Alexa 555, TdTomato, Propidium Iodide, SYTOX Orange, DyeCycle Orange
• YL2 (620/15)- PE/Texas Red, PE/AF610, Live/Dead Red, mCherry, DsRed
• YL3 (780/60)- PE/Cy7, PE/Vio770, DyeCycle Ruby

5 Laser full spectrum analzer 16UV-16V-14B-10YG-8R Coarse Wavelength Division Multiplexing (CWDM) semiconductor detector arrays for efficient spectrum capture of dyes emitting in the 365-829 nm range. High bandwidth electronics design up to 67 channels. Equipped with auto-sampler plate loader and bulk fluidics manager.

3 Laser spectral analzer 16V-14B-8R State-of-the-art optics and low-noise electronics for superb resolution of dim and rare populations. Up to 38 channels. Equipped with auto-sampler plate loader and bulk fluidics manager.

5 Laser 5UV-7V-5B-7YG-4R High-throughput smart sample handling for multi-parameter analysis.

Small particle detector upgrade with secondary forward scatter detector off of a 405nm laser. Tube or plate sample loader capable. Temperature control and sample vortexing for maintaining cell viability and clog prevention.

Call 314-362-9364 or e-mail [email protected]

From the main Barnes Jewish Hospital entrance (and the elevated walkway from the underground Barnes parking garage, aka BJC South garage), follow the South Lobby main hallway past the South Cafeteria, turn left at the corridor to the Barnard elevators (or right if you are coming from the Wohl hospital entrance), and continue past the Barnard elevators to another foyer for the Southwest Tower elevators (signs should guide you to the elevators). (Occasionally the doors that separate the two foyers are closed and appear secure. You may open and walk through them at any time to get to the Southwest Tower.) Take the elevators up to the Seventh Floor and exit the elevator foyer towards the left hallway and glass double doors. These require a card swipe, so call 314.362.9364 with the adjacent phone, and we will come and get you. Office and Synergy/Attune instruments are in SWT702, the first door on the left, and digital FACScans, FACSAria, and MoFlo are in SWT703, the second door on the left.

We are not located in the CAM building.

NOTE: If coming from the Children’s Hospital parking garage, you can use the elevated walkway to go to the Wohl stairway, then take the first floor hallway to the Southwest Tower.

Please view the table for updated pricing:

FY27 SFC Fee Schedule FINAL-1

Cancellation Policies

Analyzer appointments:

• • You have up until the start of your scheduled appointment to cancel or modify your reservation and not incur any charges. After your reservation time starts, cancelations will incur full usage fees. Shifting your time (e.g., signed up for 1-2pm and want to run 2-3pm) is acceptable at no charge as long as you will not overlap another users scheduled time. Please cancel your existing reservation prior to re-booking a new time to avoid having a cancellation fee.

Staff-operated cell sorters:

• • We kindly request that any cancellations or rescheduling of appointments be made at least 24 hours in advance. This allows us the opportunity to accommodate other clients and maintain smooth operation. Cancellations made with less than 24 hours’ notice, a late cancellation fee may apply.

• • Cancellations made with less than 24-hour notice from the start of your scheduled sort time are considered late and may incur full usage fees. If someone else schedules and uses your cancelled time, you will not be charged. If no one else schedules or uses the time slot, you will be charged full usage fees for your late cancellation. No call, no shows also incur fees for the entire reservation.

Exceptions:

• • We understand that unexpected circumstances can arise. Exceptions to the late cancellation fee may be considered for emergencies or extenuating circumstances, evaluated on a case-by-case basis. Please contact our lab as soon as possible to discuss any such situations.

Communication:

• • Cancellations or rescheduling requests can be communicated via email, phone or through iLab. Please ensure you receive confirmation of your cancellation to avoid any misunderstandings.

By adhering to this policy, you help us to better serve you and our other clients. We appreciate your understanding and cooperation.

Shared Equipment Usage and Schedule Adjustment Policy

To ensure the fair and efficient use of shared equipment and to maintain a clear and orderly scheduling system, this policy outlines the procedures for scheduling, overbooking equipment and requesting changes to the schedule.

Scheduling and Booking:

• • All shared equipment must be booked in advance by email, phone or through iLab. Staff-operated sorting request must be submitted 24 hours in advance.

• • Bookings should be made based on anticipated need and not as placeholders.

• • Users must accurately adhere to their scheduled time slots to ensure optimal utilization of equipment.

Overbooking:

• • Overbooking of flow cytometry equipment, where a user intentionally or unintentionally reserves the instrument for longer than needed, can lead to delays and inconveniences for other users. Please be mindful of how long it takes to run your samples so you can accurately schedule appointment lengths.

Requesting Schedule Changes:

• • Staff-operated sorting change requests must be submitted at least 24 hours in advance.

• • Users needing to amend scheduled analysis times after the calendar lockout period must submit a change request to SFC staff. Change requests should include the reason for the change and the amended start and stop times.

• • Upon receiving the change request SFC staff will approve or deny the request based on equipment availability and overall integrity of the request. Repeated overbooking or schedule change requests without sufficient justification may result in restricted access to the equipment.

Compliance

All users are expected to comply with this policy to ensure fair and efficient use of shared equipment. Non-compliance may result in disciplinary actions, including restricted access to the equipment.

We ask that all users maintain open and respectful communication regarding shared equipment usage.

If necessary, disputes related to equipment scheduling and usage will be mediated by SFC staff.

Our core can help plan and design experiments in regard to appropriate conjugate markers and proper process. We do not harvest, disassociate, or prepare flow cytometry samples for analysis or cell sorting. This is because of the sheer diversity of tissues and organisms dealt with, and we feel that there is mentorship available within your own specialty.

Non-adherent cells from culture, whole blood, PBMCs, bone marrow and cells from solid tissue are all acceptable samples for flow cytometry once disaggregated and red cells removed by lysis. Primary human samples or samples infected with BSL2 pathogens require special handling and notice must be given to SFC prior to sorting.

Single cells must be suspended at a density of 10-15 million cells/ml and passed through a 50um nylon mesh filter prior to sorting in order to reduce the chances of clogging the flow cytometer and its tubing. Cell concentration influences the rate of flow sorting, which can progress anywhere from 1,000–20,000 cells/second. Incorrect suspension density may lead to higher sort speeds and lower yield or recovery.

In general, cell suspensions for sorting should be brought in BD Falcon 5mL polypropylene round-bottom tubes (REF352063) with starting sample volumes no less than 300uL.

The number of cells to prepare for sorting varies greatly by experiment but is ultimately dependent on how many cells you need to recover, the frequency of the target population and % yield. To put expectations in real terms, you should prepare for the worst-case of 70% yield, which is apart from the idea of know aggregation gated by pulse width.  When you absolutely require a recovered number of cells for a downstream process, presort cell numbers should be 1.5 X target frequency.  Beginning with a surplus will assure that you will get what you need.

When sorting, we try to maintain a ratio of 6:1, nozzle size to largest cell size in suspension. This will reduce shear factors and limit stresses put on the cells during the sorting process. While most non-adherent cell lines and cell suspensions will do fine with the 70um or 100um nozzle, other sensitive cells may require larger nozzles.  Please discuss with a member of the SFC if you are unsure what to request.

For a suspension buffer, we recommend Hank’s Balanced Salt Solution (HBSS) or Phosphate Buffer Saline (PBS) with no calcium or magnesium, and no phenol red. For cell nutrition, the use of Fetal Calf Serum (FCS) or Bovine Serum Albumen (BSA) is recommended depending on the cell line, and should be between one and two percent. The use of EDTA is optional based on the cell line and its aggregation characteristics, and normally 1mM is all that is necessary.

Receiving media varies by cell type and experiment but in general should contain twenty to forty percent FCS or BSA and antibiotics (pen/strep or gentamicin). After sorting, cells should be allowed to rest for at least thirty minutes before being spun down and re-suspended in their usual medium. We can also sort directly into lysis buffer, but please remember that sorted cells are contained within a droplet of sheath and not merely cells alone. Volume is added to the collection tubes at a rate of about 2.5mL per 1 million cells with our larger 100um tip and 1mL per million cells with the 70um tip.

For proper instrument set up, controls are needed every time an experiment is done. Unstained cells, along with positive and negative controls, are needed. Positive controls are used for compensation of fluorochrome spectral overlap. Remember that a fluorochrome’s emission is measured, not in a single detector, but in all the detectors being used in the experiment. Positive controls must have an adequate signal and percent positive to adjust settings. Negative controls should consist of unstained samples and fluorescence minus one (FMO) fluorochrome staining where applicable. FMO staining should include positive staining minus one of the fluorochromes. Compensation controls are critical to the determination of what we call positive or negative for a given marker in an experimen;, they are absolutely critical to the success of the experiment.

All of the sorters are able to sort up to four populations at once. Four-way sorts must be collected into 5mL polypropylene round-bottom tubes or Eppendorf tubes, as there is a limit to the maximum distance we can separate the sort streams.

For instrument scheduling, please email [email protected]. The following information is required at the time of your request:

• Name, phone number and PI
• Cell or tissue type
• Approximate size of the largest cell in population and an estimate of the total number of presort cells
• How the cells have been treated (transfected, transduced, stimulated)
• Which fluorchromes or fluorescent proteins are being used
• Downstream applications for the cells after they have been sorted
• Number of populations to be recovered
• Bulk or plate sorting required