Clone:
H1.2F3
Type of antibody:
Primary antibodies
Isotype:
hamster IgG1
Applications:
FC
Alternative names:
AIM, VEA, EA1, MLR3, gp34/28

Extended validation for CD69 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 H1.2F3
310106++
REA937++
Cells were incubated with an excess of purified unconjugated CD69 (H1.2F3) 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 CD69. Splenocytes from C57BL/6 mice stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 24 hours were stained with CD69 antibodies and with a suitable counterstaining. As a control, CD69 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. CD4+ cells were pregated for the analysis. 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 CD69. Splenocytes from C57BL/6 mice stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 24 hours were stained with CD69 antibodies and with a suitable counterstaining. As a control, CD69 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. CD4+ cells were pregated for the analysis. 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 CD69. Splenocytes from C57BL/6 mice stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 24 hours were stained with CD69 antibodies and with a suitable counterstaining. As a control, CD69 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. CD4+ cells were pregated for the analysis. 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 CD69. Splenocytes from C57BL/6 mice stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 24 hours were stained with CD69 antibodies and with a suitable counterstaining. As a control, CD69 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. CD4+ cells were pregated for the analysis. 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 CD69. Splenocytes from C57BL/6 mice stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 24 hours were stained with CD69 antibodies and with a suitable counterstaining. As a control, CD69 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. CD4+ cells were pregated for the analysis. 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 CD69 (H1.2F3). 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 CD69 (H1.2F3). 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 CD69 (H1.2F3). 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 CD69 Antibody, anti-mouse

Overview

Clone H1-2F3 recognizes mouse CD69, a type II integral membrane protein with a C-type lectin domain. CD69 is expressed as a homodimer composed of heavily glycosylated subunits. CD69 is rapidly induced upon activation of T and B cells, neutrophils, and NK cells, which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown. Freshly prepared thymocytes undergoing selection events express CD69, and regulatory roles for CD69 expression in T-cell development in the thymus have been suggested. However, phenotypical analysis in previous studies using CD69-deficient mice has revealed that CD69 does not appear to be required for the development of CD4 T cells. CD69 is also expressed on platelets. Recent studies have shown that CD69 is constitutively expressed by tissue-resident Th memory cells and that its function is essential for the generation of professional resting memory Th cells.

Alternative names

AIM, VEA, EA1, MLR3, gp34/28

Detailed product information

Technical specifications

CloneH1.2F3
Clonalitymonoclonal
Isotypehamster IgG1
Hosthamster
Type of antibodyPrimary antibodies
Speciesmouse
AntigenCD69
Alternative names of antigenAIM, VEA, EA1, MLR3, gp34/28
Molecular mass of antigen [kDa]22
Distribution of antigenB cells, NK cells, T cells, neutrophils
Entrez Gene ID12515
RRIDAB_2659080, AB_2659081, AB_2659082, AB_2659083, AB_2659086, AB_2659087, AB_2659088, AB_2659089, AB_2659090, AB_2659091, AB_2659092, AB_2659093, AB_2659096, AB_2659097

Resources for CD69 Antibody, anti-mouse

Certificates

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to search for Certificates of Analysis (CoA) by lot number.

References for CD69 Antibody, anti-mouse

Publications

  1. Sathaliyawala, T. et al. (2013) Distribution and compartmentalization of human circulating and tissue-resident memory T cell subsets. Immunity 38(1): 187-197
  2. Shinoda, K. et al. (2012) Type II membrane protein CD69 regulates the formation of resting T-helper memory. Proc. Natl. Acad. Sci. U.S.A. 109(19): 7409-7414
  3. Sancho, D. et al. (2005) CD69 is an immunoregulatory molecule induced following activation. Trends Immunol. 26(3): 136-140

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