University of Pittsburgh Cancer Institute (UPCI)

General Guidelines

• Users must sign up for cytometer time in advance. Cancellations must be made by noon on the day before the scheduled time (this can be done by phone). Your account will be charged for the full time of a missed appointment.
• If you sign up for a time slot, please be on time. The charge ‘clock’ starts at the beginning of your scheduled appointment, even if you are not using the machine yet (unless you are waiting for the previous operator to finish). If you finish early, you will not be charged for time you did not use.
• Please finish on time. Users must yield the cytometer to the next scheduled user.

Special Guidelines for the Amnis ImageStream 100 imaging flow cytometer

• Samples must be at a very high concentration. A minimum of 20 x 106 cells per/mL is permissible, but acquisition will be slow. The optimal concentration is 50 x 106 cells/mL. The minimum cell number is 2 x 106 per sample.
• Samples must be filtered through a 70 micron cell strainer (Falcon 35-2350 or equivalent). Undoing a sample clog can take up to 2 hours on the ImageStream; you will be charged for this time if you did not filter your sample. Samples should be prepared in 0.5 mL flat top microcentrifuge tubes (Fisherbrand 05-408-128) or equivalent. Use these tubes from the start to avoid sample loss during transfer.
• Remember to prepare single-stained tubes for each fluorochrome, for color compensation. Draq-5 (Alexis Biochemicals, BOS-889-001-R200) is very useful for visualizing the nucleus. This dye occupies the FL6 channel (similar to PE-Cy5). Cells do not have to be permeabilized for Draq-5 uptake. It is not washed out and can be added 10 minutes prior to sample acquisition. Use the Amnis worksheet to design your experiment and record cytometer settings.

Special Guidelines for the MoFlo High Speed Sorter User

First time users or experienced users with a new project should contact Micheal Meyer before setting up an appointment.

Sample Requirements

• All samples must be in single cell suspension. Cells must be filtered through a <70 mm nylon mesh filter to prevent clogging the nozzle if the cell size is less than 70 μm.

• Sample may contain 1-3% FCS, or <=0.2% BSA.
• Concentration ideally should be at least 1x107/ml and can be up to 5x107/ml for non-adherent cells (low cell concentrations slow down sorting).
• Sample tube must be in a 12 x 75mm tube.
• For the purpose of compensation for multicolor analysis or sorting, you must bring an un-stained control tube plus single color stained tubes for each fluorochrome (>5% positive). Ask us about using IgG capture beads (BD CompBeads, Cat # 552843) instead of cells for single stained controls.
• No radioactive material. The facility does except unfixed human or non-human primate lenti virus-transfected cells, but investigators must meet with and discuss these experiments with facility staff. Please make an appropriate appointment if you need to perform a biohazardous sort.

Collection Container (User supplies one of following options)

• Tubes: 12 x 75mm, 1.5ml, 15ml or 50ml.
• Plates:n24, 96 (include PCR plate), 384 well or custom size.
• Trays/dishes or slides.
• Fill collection container (~10% of volume) with desired collection medium.

Special Guidelines for Coulter EPICS XL Users

• All individuals desiring to operate the User XL must complete training (‘Basic’ or ‘Power User’) with a member of the UPCI Flow Cytometry Facility. The five-day basic course offered by Beckman-Coulter will count as “Power User” training.
• Basic users are limited to cytometer operation between the hours of 9 a.m. and 5 p.m., Monday through Friday. “Power Users” designated by the Facility may sign up to use the cytometer after hours and on holidays and weekends. Basic users can use the cytometer after hours only if supervised by a “Power User”.
• Please keep the work area clean.
• Proper procedures must be followed concerning sample acquisition, instrument cleaning, and data management/backup. For assistance, please refer to the UPCI Flow Facility Survival Guide, summary sheets located by the cytometer, and facility personnel.
• All operators using the cytometer must properly fill out the time and effort log, grant account sheets, and data file list.
• The Facility reserves the right to revoke privileges from individuals who consistently disregard these policies.
• The recommended sample volume is no less than 0.3 ml.
• Samples should be resuspended in 12 X 75 mm FALCON 5 ml tubes (Falcon # 352002 and 352063). Slightly undersized tubes will damage the sample head. If you think your tube may not fit properly please ask us for the tubes to transfer your sample.
• Samples should be fixed in 0.5-2% paraformaldehyde or methanol free formaldehyde to kill infectious material. Do not use common laboratory grade formaldehyde. Infectious material should be fixed in paraformaldehyde for at least 30 minutes for best results. If cells cannot be fixed, please make prior arrangements with the Facility.
• Please, no unfixed infectious material or radioactive material. Filter cells that are sticky or clumping with nylon mesh (Falcon 5ml Polystyrene Round Bottom Tube with Cell-Strainer Cap 12x75mm; Part number 352235) or 1cc Tuberculin syringes. Anything less than 70 microns should flow through the cytometers. However, this assumes a single cell suspension. Cells that grow in monolayers, as well as large populations of dead cells or debris will cause samples to clump.