CD275 (B7-H2) Antibody, anti-mouse

Name: CD275 (B7-H2) Antibody, anti-mouse
Availability: InStock

CD275 (B7-H2) Antibody, anti-mouseExtended Validation

Clone:

HK5.3

Type of antibody:

Primary antibodies, Functional-grade antibodies

Isotype:

rat IgG2aκ

Applications:

FA, FC

Alternative names:

ICOSL, B7-H2, B7RP-1, B7h, ICOSLG, LICOS, Ly115l, GL50, GL50-B

Flow cytometry applications forCD275 (B7-H2) Antibody, anti-mouse

APC
Biotin
PE
PE-Vio 770

Conjugate:

APC

Recommended dilution:

1:10

Remark:

Cells should be stained prior to fixation, if formaldehyde is used as a fixative.

Tested:

QC tested

Products used:

130-102-944, 130-102-247

Protocols:

Cell surface flow cytometry staining protocol (PBS/EDTA/BSA 1:10)

Description:

Untouched B cells isolated from BALB/c mouse splenocytes were cultured overnight and stained with CD275 (B7-H2) antibodies conjugated to PE, APC, PE-Vio770 and Biotin, as well as with CD45R (B220)-APC or CD45R (B220)-PE and analyzed by flow cytometry using the MACSQuant® Analyzer. As control (left images), unseparated mouse splenocytes were stained with CD275 antibodies conjugated to fluorochromes. Cells labeled with CD275 (B7-H2)-Biotin were stained with Anti-Biotin-PE as well. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of CD275 (B7-H2)-PE-Vio770.

Functional assay applications forCD275 (B7-H2) Antibody, anti-mouse

pure-functional grade

Conjugate:

pure-functional grade

Tested:

during development

Products used:

130-106-227

Type of functional assay:

blocking of receptor-ligand binding

Extended validation for CD275 (B7-H2) Antibody, anti-mouse

Specificity

Epitope competition

In order to compare the epitope specificity of an antibody, the clone being used is compared with other known clones recognizing the same antigen in a competition assay.

Other clones	Overlap in epitope recognition with HK5.3
REA990	++
Cells were incubated with an excess of purified unconjugated CD275 (B7-H2) (HK5.3) antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker. Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.

Sensitivity

Performance comparison

Selected fluorochrome conjugated antibodies from Miltenyi Biotec were compared to commercially available hybridoma clones in flow cytometry analysis.

Flow cytometric comparison of different clones for CD275 (B7-H2). Mix of unseparated C57BL/6 Mouse Splenocytes and isolated B-cells stored overnight were stained with CD275 (B7-H2) antibodies and with a suitable counterstaining. As a control, CD275 (B7-H2) antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for CD275 (B7-H2). Mix of unseparated C57BL/6 Mouse Splenocytes and isolated B-cells stored overnight were stained with CD275 (B7-H2) antibodies and with a suitable counterstaining. As a control, CD275 (B7-H2) antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for CD275 (B7-H2). Mix of unseparated C57BL/6 Mouse Splenocytes and isolated B-cells stored overnight were stained with CD275 (B7-H2) antibodies and with a suitable counterstaining. As a control, CD275 (B7-H2) antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for CD275 (B7-H2). Mix of unseparated C57BL/6 Mouse Splenocytes and isolated B-cells stored overnight were stained with CD275 (B7-H2) antibodies and with a suitable counterstaining. As a control, CD275 (B7-H2) antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for CD275 (B7-H2). Mix of unseparated C57BL/6 Mouse Splenocytes and isolated B-cells stored overnight were stained with CD275 (B7-H2) antibodies and with a suitable counterstaining. As a control, CD275 (B7-H2) antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Fixation data

To provide an indication on how an antibody performs after fixation of cells, in-house data on staining results before and after fixation with 3.7% formaldehyde using Miltenyi Biotec antibodies are provided. Different experimental settings may lead to different results.

No fixation

Post fixation

Comparison of staining pattern on non-fixed and fixed cells using CD275 (B7-H2) (HK5.3). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.

Comparison of staining pattern on non-fixed and fixed cells using CD275 (B7-H2) (HK5.3). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.

Specifications for CD275 (B7-H2) Antibody, anti-mouse

Overview

Clone HK5.3 recognises murine CD275, which is also known as B7-H2 and ICOSL. CD275 is a type I membrane protein and belongs to the Ig superfamily, B7 family of costimulatory molecules. Expression of CD275 is found on antigen presenting cells such as B cells, dendritic cells, and monocytes. CD275 costimulates T cells upon binding to ICOS, which is expressed on activated T cells. In mouse, CD275 binds only ICOS, whereas in humans it can interact with other T cell costimulatory molecules, CD28 and CTLA-4. To appropriately regulate the activation of immune response, the ICOS-CD275 interaction can also lead to downregulation of CD275 expression which allows prevention of hyperreaction of T cells.

Alternative names

ICOSL, B7-H2, B7RP-1, B7h, ICOSLG, LICOS, Ly115l, GL50, GL50-B

Detailed product information

Technical specifications

Clone	HK5.3
Clonality	monoclonal
Isotype	rat IgG2aκ
Isotype control	Isotype Control Antibody, rat IgG2a
Host	rat
Type of antibody	Primary antibodies, Functional-grade antibodies
Species	mouse
Antigen	CD275 (B7-H2)
Alternative names of antigen	ICOSL, B7-H2, B7RP-1, B7h, ICOSLG, LICOS, Ly115l, GL50, GL50-B
Molecular mass of antigen [kDa]	31
Distribution of antigen	dendritic cells, macrophages, monocytes
Entrez Gene ID	50723
RRID	AB_2656897, AB_2656898, AB_2656899, AB_2656900, AB_2656901, AB_2656902, AB_2656903, AB_2656904, AB_2656886

Resources for CD275 (B7-H2) Antibody, anti-mouse

Certificates

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References for CD275 (B7-H2) Antibody, anti-mouse

Publications

Watanabe, M. et al. (2008) Down-regulation of ICOS ligand by interaction with ICOS functions as a regulatory mechanism for immune responses. J Immunol 180: 5222-5234
Yao, S. et al. (2011) B7-H2 is a costimulatory ligand for CD28 in human. Immunity 34(5): 729-740

Applications

Extended validation