Binding Buffer:

• 10mM HEPES/NaOH, pH 7.4
• 150mM NaCl
• 5mM KCl
• 1mM MgCl₂
• 1.8mM CaCl₂

PI Solution:

• Propidium iodide 100µg/ml in Binding Buffer

1. 1Wash 2x10⁶ cells with PBS.
2. Dilute FITC-Annexin V to 1µg/ml in binding buffer and resuspend cells in 1ml of this solution (prepared fresh each time)
3. Incubate 10 min. in the dark at room temperature
4. Add 10µl of PI solution for a final concentration of 1µg/ml prior to analysis

1. Wash cells in PBS and resuspend 2 x 10⁶ cells in 0.5 ml PBS.
2. Fix cells by addition of 5 ml of 1% paraformaldehyde. Incubate on ice for 15 min.
3. Wash cells twice and resuspend in 0.5 ml PBS in polypropylene tubes.
4. Permeabilize cells by addition of 3 ml of ice-cold ethanol (70%) for at least 4 hr on ice.
   (Cells may be stored for several weeks in 70% ethanol at -20^oC)
5. Centrifuge sample, wash in PBS and resuspend cells in 50 µl of TdT reaction solution:
   • 10 µl TdT reaction buffer
   • 2.0 µl BrdUTP stock solution
   • 15 U TdT, 5µl CoCL2 (10 mM)
   • 33.5 µl H₂0
6. Incubate cells for 40 min at 37^oC with occasional mixing (or room temp. overnight).
7. Add 1.5 ml rinsing buffer and centrifuge.
8. Add anti-BrdU-FITC antibody (approx. 100 µl) and incubate at room temp. for 1 hr.
9. Add 1ml of PI staining solution, incubate for at least 30 min at room temp. in the dark.
10. Analyze for FITC/PI fluorescence with appropriate controls for instrument setup.

The exact incubation times for fixation, permeabilization, TdT may vary according to cell type. It may be necessary to optimize these for specific cells and applications

The exact incubation times for fixation, permeabilization, TdT may vary according to cell type. It may be necessary to optimize these for specific cells and applications.

Reagents:

TdT Reaction Buffer (5X):

• 1M potassium or sodium cacodylate, 125mM TRIS-HCl, pH 6.6, 1.25mg/ml BSA. Store at -20ºC.

Anti-BrdU-FITC Antibody Solution:

• 0.3µg anti-BrdU-FITC antibody, 0.3% w/v Triton X-100, 1% BSA, PBS to 100µl volume per test.

PI Staining solution:

• Propidium Iodide 5µg/m PI, RNase A (DNase free) 200µg/ml, PBS, pH 7.2-7.4

Many vendors sell kits with pre-prepared buffers and reagents. Alternatively, individual reagents may be purchased separately. The above protocol is a general guide and individual kits may vary in the amounts of reagents used.

1. Add 1µl of 1 mg/ml 7-AAD (1µg/ml final) to approximately 1x10⁶ washed cells suspended in 1ml PBS.
2. Incubate ≥ 30 minutes in the dark on ice.
3. Analyze on flow cytometer with excitation at 488nm. Collect fluorescence emission with a 650nm long-pass or a 670±20nm band-pass filter.

7-AAD is easily excitable at 488nm. The fluorescence emission of the dye has a peak at ~670nm.

From Current Protocols in Flow Cytometry

We would appreciate it if the Cytometry Core Facility is mentioned in the acknowledgement section of any publication which includes results of data produced from the facility, and that an electronic copy of the paper be provided to the facility. This will help us justify upgrading services and equipment on grant applications.

Please cite the facility as: "UACC/ARL Cytometry Core Facility" and include the Cancer Center Support Grant (CCSG - CA 023074)

Methods Section of the Paper

Please use the links below to create the configuration specific to your application. Following is an example of a typical description. Please contact the lab personnel if you wish assistance with the wording:

Two-color flow cytometric analysis was performed using a FACScan flow cytometer (BDBiosciences, San Jose, CA) equipped with an air-cooled 15mW argon ion laser tuned to 488nm. The emission fluorescence of (yada, yada conjugated to IgG...blah blah...) was detected and recorded through a 530/30 bandpass filter in the FL1 channel. (Something red) was detected in the FL2 channel through a 585/42 bandpass filter. List mode data files consisting of 10,000 events gated on FSC (forward scatter) vs SSC (side scatter) were acquired and analyzed using CellQuest PRO software (BDBiosciences, San Jose, CA) at a rate of 200-400 events per second. Appropriate electronic compensation was adjusted by acquiring the cell populations stained with each dye/fluorophore individually, as well as an unstained control.

We provide personalized services. A consultation is required before the first scheduled session to discuss controls needed and to expedite any special requests. This can easily be accomplished by contacting the Facility Manager.

An account or grant number must be included with each sample run. Charges are based on 15 minute increments for analysis or sorting. It is the responsibility of each user to keep his/her User Profile updated regarding account information, phone number, and email address.

Because of the complexity of the FACSAria, there is a one-hour setup charge for each sorting session.

If you are not affiliated with the Arizona Universities, please contact the Cytometry Core Facility for an initial consultation and service quotes.

See Rates

The FACSAria has a jet-in-air fluidics system, which potentially produces aerosols, especially when sorting. These aerosols may contain infectious agents, such as hepatitis or HIV, when sourced from live human tissue. In addition, sorting known infectious agents such as bacteria or cells transfected or transformed with infectious agents also pose a risk from aerosol exposure. Since this aerosol is under pressure, potential for infectivity is theoretically much higher than incidental exposure during analysis.

The addition of a Baker BioProtect III biosafety cabinet, in which the FACSAria is now housed, allows for sorting of certain live human tissue and BSL2 agents. Approval for BSL2 sorting must be obtained through the Core Facility Manager.

Turn-around time is dependent on the length of the appointment. In general, samples are processed and data is received by the user at the end of their appointment time.

Samples for the FACScanto II and the LSR II must be provided in 12x75mm polystyrene BD brand Falcon tubes. The only tubes compatible with these analyzers are:

• Cat. # 35-2052 - sterile, no cap, 125/bag
• Cat. # 35-2054 - sterile, with cap, 125/bag
• Cat. # 35-2058 - sterile, with cap, 25/bag
• Cat. # 35-2008 - nonsterile, no cap, 1000 bulk
• Cat. # 35-2235 - sterile, with cell strainer, 25/bag

Samples for the FACSAria may be provided in 12x75mm polystyrene tubes or 15ml conical tubes.

The recommended sample concentration for the FACScanto II and the LSR II is 1x10⁶ cells per ml in PBS (no Mg⁺⁺, Ca⁺⁺ or serum) or fixative.

1-3% paraformaldehyde is the recommended fixative for most cell types.

The recommended sample concentration for the FACSAria is 3x10⁶-5x10⁶ cells per ml in PBS (no Mg⁺⁺, Ca⁺⁺ or serum) or fixative.

Pathogenic samples or live samples derived from human sources must be approved by the Facility Manager. When bringing samples for multi-parameter analysis, single positive controls must be provided for each color user.

Cell suspensions are typically prepared in the investigator's laboratory and brought to the Cytometry Core Facility ready to be run. There is a wet lab available in the facility with limited equipment for use in the preparation of samples, if needed.