FITC Mouse Anti-Human CD3ε
Clone APA1/1 (RUO)
- Brand BD Pharmingen™
- Vol. Per Test 20 µl
- Isotype Mouse IgG1, κ
- Reactivity Human (QC Testing)
Intracellular staining (flow cytometry) (Routinely Tested)
- Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
Monoclonal antibody APA1/1 reacts with the intracellular domain of the epsilon chain of the CD3 molecule (CD3-ε). This antibody does not react with the extracellular region of CD3-ε. The assembly of the T cell antigen receptor (TCR)-CD3 complex has been suggested to take place by pairwise interactions of the CD3-ε subunit with either CD3-γ or CD3-δ. These dimers then associate with the TCR heterodimer a/β or γ/δ, and the CD3-ζ homodimer to form the full complex. Studies show that antibodies APA1/1 and SP34 gave a strong reaction with COS cells singly transfected with CD3-ε. Other anti-CD3 antibodies (OKT3, WT31, UCHT1, Leu4) did not react with COS cells singly transfected with CD3-ε. This reagent could be useful for the study of T cell development or the study of conformational changes of CD3-ε upon ligand binding to TCR-CD3 complex.
FITC, fluorescein isothiocyanate, is a fluorochrome with a molecular weight of 389 Da. FITC is sensitive to pH changes and photobleaching. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor® 488 cannot be used simultaneously. FITC is relatively dim and should be reserved for highly expressed markers whenever possible.
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Preparation and Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
- 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).
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
Protocol for PBMC cells fixation, permeabilization and staining
1. Harvest, count and pellet PBMC cells following standard procedures.
2. While vortexing, add 5 ml cold 70% - 80% ethanol dropwise into the cell pellet (1-5 x 10^7 cells). Incubate at -20°C for at least 2 hours. These fixed cells can be stored at -20°C for up to 60 days prior to staining.
3. Wash twice with 30-40 ml staining buffer (PBS with 1% FBS, 0.09% NaN3), centrifuge for 10 minutes at 200 x g.
4. Resuspend the cells to a concentration of 1 x 10^7/ml.
5. Transfer 100 µl (1 x 10^6 cells) cell suspension into each sample tube.
6. Add 20 µl of properly diluted fluorescence conjugated antibody into the tubes above. Mix gently.
7. Incubate the tubes at room temperature (RT) for 20-30 minutes in the dark.
8. Wash with 2 ml of staining buffer at 200 x g for 5 minutes.
9. Aspirate the supernatant.
10. Add 0.5 ml of staining buffer to each tube.
11. Proceed to flow cytometric analysis.