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

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 clonesOverlap 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.
View details
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.
View details
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.
View details
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.
View details
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.
View details
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
View details
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.
View details
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

CloneHK5.3
Clonalitymonoclonal
Isotyperat IgG2aκ
Isotype controlIsotype Control Antibody, rat IgG2a
Hostrat
Type of antibodyPrimary antibodies, Functional-grade antibodies
Speciesmouse
AntigenCD275 (B7-H2)
Alternative names of antigenICOSL, B7-H2, B7RP-1, B7h, ICOSLG, LICOS, Ly115l, GL50, GL50-B
Molecular mass of antigen [kDa]31
Distribution of antigendendritic cells, macrophages, monocytes
Entrez Gene ID50723
RRIDAB_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

Please follow this
link
to search for Certificates of Analysis (CoA) by lot number.

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

Publications

  1. 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
  2. Yao, S. et al. (2011) B7-H2 is a costimulatory ligand for CD28 in human. Immunity 34(5): 729-740

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