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FITC Mouse Anti-Rat IgG1
FITC Mouse Anti-Rat IgG1

Detection of intracellular rat IgG1 in an antibody-secreting hybridoma cell line. Cells were fixed, permeabilized, and stained according to the method described below using FITC-conjugated RG11/39.4 mAb (filled histogram) or the matched isotype control, FITC-conjugated 27-35 mAb (open histogram, Cat. No. 555057). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Detection of intracellular rat IgG1 in an antibody-secreting hybridoma cell line. Cells were fixed, permeabilized, and stained according to the method described below using FITC-conjugated RG11/39.4 mAb (filled histogram) or the matched isotype control, FITC-conjugated 27-35 mAb (open histogram, Cat. No. 555057). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Pharmingen™
Rat (QC Testing)
Mouse SJL IgG2a, κ
Pooled rat IgG1
Flow cytometry (Routinely Tested), Intracellular staining (flow cytometry) (Tested During Development)
0.5 mg/ml
AB_395120
Aqueous buffered solution containing protein stabilizer and ≤0.09% sodium azide.
RUO


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.

Recommended Assay Procedures

RG11/39.4 antibody is effective for detection of cell-surface or intracellular Ig by immunofluorescent staining with flow cytometric analysis. FITC-conjugated RG11/39.4 mAb may be used as a primary or secondary reagent in immunofluorescent staining.

Immunofluorescent Staining of Intracellular Immunoglobulin (Ig) Protocol

1.   Prepare a single-cell suspension and determine cell number.

2.   Suspend cells in staining buffer (PBS + 2% FBS + 0.1% Sodium Azide) at 2 × 10^7 cells/ml and transfer to U-bottom microwell plates in 50 µl/well for immunofluorescent staining.

Note: The BD Pharmingen™ Stain Buffer with FBS (Cat. No. 554656) is effective for use as a staining buffer in this protocol.

3.   Block Fcγ receptors by adding 0.2 µg of purified 2.4G2 antibody (Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2) (Cat. No. 553141/553142) 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 surface Ig with purified RG11/39.4 mAb (Cat. No. 553889) 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™ intracellular staining buffer (BD Cytofix/Cytoperm™ Kit, Cat. No. 554714) per well.

10. Incubate 30 minutes at room temperature.

11. Wash 2× with 200 µl of 1× BD Perm/Wash™ buffer (provided in the BD Cytofix/Cytoperm Kit) per well. Centrifuge at 250 × g

for 5 minutes and aspirate supernatant between washes.

12. Stain intracellular Ig by adding ≤ 1 µg of FITC-conjugated RG11/39.4 mAb 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.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. 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.
553892 Rev. 2
Antibody Details
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RG11/39.4

The RG11/39.4 antibody reacts specifically with the Fc region of rat IgG1. It does not react with other Ig isotypes.

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.

553892 Rev. 2
Format Details
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FITC
Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
FITC
Blue 488 nm
494 nm
518 nm
553892 Rev.2
Citations & References
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Development References (1)

  1. Springer TA, Bhattacharya A, Cardoza JT, Sanchez-Madrid F. Monoclonal antibodies specific for rat IgG1, IgG2a, and IgG2b subclasses, and kappa chain monotypic and allotypic determinants: reagents for use with rat monoclonal antibodies. Hybridoma. 1982; 1(3):257-273. (Immunogen). View Reference
553892 Rev. 2

Please refer to Support Documents for Quality Certificates


Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.

Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.