BD Horizon™ V450 Mouse Anti-Human FoxP3

Cell Preparation and Staining Procedures for Conjugated Anti-Human FoxP3 Antibody

          1.  Bring the buffers to RT before use. Prepare working solutions of the BD Pharmingen Human FoxP3 Buffer Set

               Cat. No. 560098 (For the buffer preparation, please see TDS Cat. No. 560098 buffer instructions for details).

          2.  Prepare human PBMC. Dilute the cells with BD Pharmingen Stain Buffer (FBS)* to ten million cells/ml.

          3.  Pipette appropriate amount of surface staining reagent to bottom of each 12 x 75 mm tube.

          4.  Add 100µl of cells per tube, vortex, incubate for 20 minutes at RT protected from light.

          5.  Add 2 ml of wash buffer. Centrifuge 250 x g for 10 minutes, and remove wash buffer.

          6.  To fix the cells, gently re-suspend pellet in residual volume of wash buffer and then add 2ml of 1x Human FoxP3 Buffer A. Vortex.

                Incubate for 10 minutes at RT in the dark.

          7.  Centrifuge 500 x g for 5 minutes, and remove fixative. Caution: Be aware the pellet is buoyant.

          8.  To wash cells, re-suspend each pellet in 2ml of BD Pharmingen Stain Buffer (FBS)*, and centrifuge 500 x g for 5 minutes. Remove

               wash buffer.

          9.  To permeabilize the cells, gently re-suspend pellet in residual volume of wash buffer and then add 0.5 ml of 1x working solution

               Human FoxP3 Buffer C to each tube. Vortex.  Incubate for 30 minutes at RT protected from light.

        10.  To wash cells, add 2 ml of BD Pharmingen Stain Buffer (FBS)* to each tube, centrifuge 500 x g for 5 minutes at RT.  Remove buffer

                and repeat wash step.  Remove buffer.

        11.  Add conjugated FoxP3 antibody at appropriate concentrations to re-suspend the pellet. Gently shake or vortex.

        12.  Incubate for 30 minutes in the dark at RT.

        13.  Repeat wash step #10.

        14.  Resuspend in wash buffer and analyze immediately.

                Optional  Add 300µl of 1% formaldehyde in 1x PBS and store at 4°C.  Analyze cells within 24 hours.

*   We recommend using the BD Pharmingen Stain Buffer (FBS; Cat No. 554656) for all wash steps and covering tubes during incubation steps with caps or parafilm.  We also recommend optimizing forward scatter and side scatter voltages to visualize lymphocytes as separate from debris, red cell ghosts and/or platelets before acquisition.

**  Acquire at least 15,000 to 25,000 CD4 positive lymphocytes.

1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
2. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
5. BD Horizon V450 has a maximum absorption of 406 nm and maximum emission of 450 nm. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
6. Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
7. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.