Mouse Foxp3 Fixation Concentrate (20x) (component 51-9006124):10 mL. Store at 4°C and protect from prolonged exposure to light. This is a 20× stock solution that must be diluted prior to use with normal 1× PBS without FBS.Preparation of 1× Fixation Buffer: Dilute 1 part Fixation Concentrate with 19 parts 1× PBS. Prepare 2 mLfor each test (1x10^6 cells). Put on ice.Do not store 1× Fixation Buffer, prepare fresh every time.

Mouse Foxp3 Permeabilization Concentrate (5x) (component 51-9006125):80 mL. Store at 4°C. This is a 5× stock solution that must be diluted prior to use with normal 1× PBS without FBS.Preparation of 1 × Permeabilization Buffer: Dilute 1 part Permeabiliation Concentrate with 4 parts 1× PBS. Prepare 4 mLfor each test (1x10^6 cells). Pre-warm to 37°C before use.Do not store 1 x Permeabilization Buffer, prepare fresh every time.

Cell Preparation and Staining Procedures for Conjugated Anti-Mouse Foxp3 Antibodies

1. Prepare a single-cell suspension from the peripheral lymphoid tissue of interest. Remove clumps of cells and/or debris by passing the suspension through a Falcon® 70-µm nylon cell strainer (Corning Cat. No. 352235). Use 1× BD PharmLyse™ Lysing Buffer (Cat. No. 555899) to lyse red blood cells if necessary. Dilute the cells with BD Pharmingen™ Stain Buffer (FBS, Cat. No. 554656) to 10 million cells/mL.

2. Pipette appropriate amount of CD4 or other surface staining reagent(s) to bottom of each 12 x 75 mm tube.

3. Add 100 µL of cells per tube, mix well. Incubate for 20 minutes at RT in the dark.

4. To wash cells, add 2 mL of BD Pharmingen™ Stain Buffer (FBS) to each tube. Centrifuge 250 x g for 10 minutes, and remove buffer.

5. To fix the cells, gently re-suspend pellet in residual volume of staining buffer and then add 2 ml of freshly prepared cold 1× BD Pharmingen™ Mouse Foxp3 Fixation Buffer. Mix well. Incubate for 30 minutes at 4°C in the dark.

6. Centrifuge 500 x g for 5 minutes, and remove fixative.

7. To wash cells, re-suspend each pellet in 2 mL of freshly prepared pre-warmed 1× BD Pharmingen™ Mouse Foxp3 Permeabilization buffer, and centrifuge 500 x g for 5 minutes. Remove permeabilization buffer.

8. To permeabilize the cells, gently re-suspend pellet in another 2 ml of freshly prepared pre-warmed 1 × BD Pharmingen™ Mouse Foxp3 Permeabilization buffer. Incubate for 30 minutes at 37°C in the dark.

9. Centrifuge 500 x g for 5 minutes, and remove buffer.

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

11. Add 20 µL of conjugated Foxp3 antibody diluted with BD Pharmingen™ Stain Buffer (FBS) at appropriate concentrations (check the figure legend from each format for the concentration) to re-suspend the pellet. Gently shake or vortex briefly.

12. Incubate for 20 minutes at RT in the dark.

13. Repeat wash step# 10 two times.

14. Resuspend the cells in 0.5 mL BD Pharmingen™ Stain Buffer (FBS) and analyze immediately.Acquire at least 15,000 to 25,000 CD4 positive lymphocytes.

Note:The BD Pharmingen™ Stain Buffer is recommended for all wash steps and covering tubes during incubation steps with caps or parafilm.Optimizing forward scatter and side scatter voltages is also recommended to visualize lymphocytes as separate from debris, red cells, etc. before acquisition.

Danger:Mouse Foxp3 Fixation Concentrate (20x) (component 51-9006124) contains 4.2% formaldehyde (w/w).

Hazard statements

Harmful if inhaled.

Causes skin irritation.

Causes serious eye damage.

May cause an allergic skin reaction.

Suspected of causing genetic defects.

May cause cancer. Route of exposure: Inhalative.

May cause respiratory irritation.

Precautionary statements

Wear protective clothing / eye protection.

Wear protective gloves.

Do not breathe mist/vapours/spray.

IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.

If skin irritation or rash occurs: Get medical advice/attention.