FITC Rat Anti-Mouse IgM
Clone II/41 (RUO)
- Brand BD Pharmingen™
- Concentration 0.5 mg/ml
- Isotype Rat IgG2a, κ
- Reactivity Mouse (QC Testing)
Flow cytometry (Routinely Tested)
Intracellular staining (flow cytometry) (Tested During Development)
- Immunogen Not reported
- Entrez Gene ID 16019
- Storage Buffer Aqueous buffered solution containing ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
The II/41 clone has been reported to react specifically with mouse IgM of Igh-C[a] and Igh-C[b] haplotypes. It has been reported not to react with other Ig isotypes. In addition, the II/41 clone has been reported not to stimulate B-cell proliferation.
This antibody is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
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.
- 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.
Recommended Assay Procedure:
Immunofluorescent Staining of Mouse Intracytoplasmic/Intracellular IgM:
1. Prepare a single-cell suspension and determine cell number.
2. Suspend cells at 2×10e7 cells/ml in BD Pharmingen™ Stain Buffer (Cat. No. 554656) [or alternatively, prepare a staining buffer made up in PBS + 2% FBS + 0.1% sodium azide] and transfer to U-bottom microwell plates in 50 µl/well for immunofluorescent staining.
3. Block Fcγ receptors by adding 0.2 µg of BD Fc Block™ (Cat. no. 553141) in 50 µl of staining buffer to each well.
4. Incubate 5 minutes on ice.
5. Add 200 µl of staining buffer/well and resuspend cells. Centrifuge at 250×g for 5 minutes and aspirate supernatant.
6. Block cell surface IgM with purified rat anti-mouse IgM (clone II/41) (Cat. No. 553435) by adding 1.0 µg per sample in 50 µl of staining buffer/well.
Note: Surface markers may be stained during this step as described in the "Immunofluorescent Staining of Mouse and Rat Leukocytes for Flow Cytometry" in the Technical Protocols section of our web site at http://www.bdbiosciences.com/pharmingen/protocols/Mouse_and_Rat_Leukocytes.shtml
7. Incubate 15 minutes on ice.
8. Wash 2× as described in Step 5.
9. Resuspend cells in 100 µl of BD Cytofix/Cytoperm™ solution (Cat. No. 554714) per well.
10. Incubate 30 minutes at room temperature.
11. Wash 2× with 200 µl of 1× BD Perm/Wash™ buffer (Cat.No. 554714) per well. Centrifuge at 250×g for 5 minutes and aspirate supernatant between washes.
12. Stain for intracellular IgM by adding ≤ 1 µg of FITC rat anti-mouse IgM (clone II/41) antibody in 50 µl of 1× BD Perm/Wash™ buffer/well.
Note: Other antibodies recommended for staining of intracellular markers may be added during this step as described in Step 12.
13. Incubate for 30 minutes at room temperature.
14. Wash 2× as described in Step 11.
15. Resuspend and transfer samples in 100 µl of staining buffer to tubes appropriate for analysis with a flow cytometer. Bring volume in each tube to 400 µl with staining buffer.
16. Analyze samples on a flow cytometer.