Foxp3/Transcription Factor Staining Kit

Background

Foxp3 / Transcription Factor Staining Kit has been formulated and optimized for staining with antibodies to transcription factors and nuclear proteins, such as Foxp3 and STAT3, as well as cytokines and chemokines.

Components

Instructions

Dilute Fixation Concentrate (4x) with Fixation Dilution Solution to 1x Fixation Working Solution before use. For example, take 1 mL Fixation Concentrate (4x) and add it to 3 mL Fixation Dilution Solution to get 4 mL 1x Fixation Working Solution. Each sample requires 1 mL of 1x Fixation Working Solution.

Dilute Permeabilization Buffer (10x) with ddH2O to 1xPermeabilization Working Solution before use. For example, take 1 mL Permeabilization Buffer (10x), and add it to 9 mL ddH2O to get 10 mL 1x Permeabilization Working Solution. Each sample requires 8.5 mL of 1x Permeabilization Working Solution.

1. Add the single-cell suspension into tubes, 1x 106 cells in 100 µL suspension per tube.
2. Stain cells with a Fixable Viability Dye. (Optional)
3. Stain cell surface markers.
4. After incubating with the cell surface marker, add 1 mL of Cell Staining Buffer, centrifuge samples at 300 g for 5 min, discard the supernatant, then resuspend the cells with 100 µL of Cell Staining Buffer.
5. Add 1 mL of 1x Fixation Working Solution to each tube and mix fully, incubate the cells at 4°C for 30 min, then centrifuge at 600 g for 5 min and discard the supernatant.
6. Add 2 mL of 1x Permeabilization Working Solution to each tube and mix fully, centrifuge at 600 g for 5 min and discard the supernatant.
7. Repeat Step 6.
8. Resuspend the cells with 100 µL of 1x Permeabilization Working Solution.
9. Block with 2 % normal mouse/rat serum by adding 2 µL directly to the cells. Incubate for 15 min at room temperature. (Optional)
10. Without washing, add the recommended amount of directly FCM antibody for detection of intracellular antigen(s) to cells and incubate for at least 30 min at room temperature in the dark.
11. Add 2 mL of 1x Permeabilization Working Solution to each tube and centrifuge at 600 g for 5 min at room temperature. Discard the supernatant.
12. Resuspend the cells with appropriate Cell Staining Buffer, then analyze the samples by flow cytometer.

Storage

Store at 4°C for six months in the dark. Avoid freeze / thaw cycles.

Cautions

1. For maximal assay performance, this reagent should be used within 6 months. Avoid freeze / thaw cycles.
2. The fixation and permeabilization steps that are required for the detection of intracellular antigens may alter the light scatter properties of cells and may increase non-specific background staining. Including extra proteins such as BSA or fetal calf serum (FCS) in the staining buffer may help reduce non-specific background. The use of Fixable Viability Dyes is recommended to help eliminate dead cells during the analysis.
3. For your safety and health, please wear the lab coat and disposable gloves before the experiments.