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90283
Rat (LTF-2) mAb IgG2b Isotype Control (redFluor™ 710 Conjugate)
Antibody Conjugates

Rat (LTF-2) mAb IgG2b Isotype Control (redFluor™ 710 Conjugate) #90283

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Flow cytometric analysis of live mouse splenocytes using MHC Class II (I-A/I-E) (M5/114.15.2) Rat mAb (redFluor™ 710 Conjugate) #29027 (solid line) compared to concentration-matched Rat (LTF-2) mAb IgG2b Isotype Control (redFluor™ 710 Conjugate) (dashed line).

To Purchase # 90283S
Product # Size Price
90283S
100 µg $ 159

Supporting Data

Concentration 200 ug/ml
SENSITIVITY
Isotype Rat IgG2b

Product Description

This Cell Signaling Technology antibody is conjugated to redFluor™ 710 under optimal conditions and tested in-house for direct flow cytometric analysis in mouse cells.

Product Usage Information

Note: This control antibody must be diluted to the same concentration (not dilution) as the specific antibody in analysis. See Directions for Use.

Directions for Use: Important: Dilute this control antibody to the same concentration (not dilution) as specific antibody in analysis. Higher background fluorescence may result if excessive amounts of rat IgG isotype control are used.

Storage:

Supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH7.2. This product is stable for 6 months when stored at 4ºC. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Flow Cytometry, Live Cell Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water, mix.
  2. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to Immunostaining.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Pellet cells by centrifugation and remove supernatant.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 30 min to 1 hr on ice. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.

posted June 2017

revised August 2019

Protocol Id: 1504

Source / Purification

The antibody is not directed against any known antigen. It functions as an isotype control for rat monoclonal antibodies.

Background

Isotype control antibodies are used to estimate the nonspecific binding of target primary antibodies due to Fc receptor binding or other protein-protein interactions. An isotype control antibody should have the same immunoglobulin type and be used at the same concentration as the test antibody.

For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
redFluor is a registered trademark of Tonbo Biosciences.