Anti-CD99 antibody [EPR3096]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD99 antibody [EPR3096] (AB108297)

Immunocytochemistry/Immunofluorescence analysis of Jurkat (Human T cell leukemia cell line from peripheral blood) labelling CD99 with purified ab108297 at 1/500. Cells were fixed with 4% PFA and permeabilized with 0.1% triton X-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

• Flow Cyt

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Flow Cytometry - Anti-CD99 antibody [EPR3096] (AB108297)

Flow cytometric analysis of Molt-4 cells using anti-CD99 RabMAb (red) (ab108297) or a rabbit IgG (negative) (green).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD99 antibody [EPR3096] (AB108297)

Immunohistochemical staining of paraffin-embedded Human pancreas tissue using ab108297 at a dilution of 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD99 antibody [EPR3096] (AB108297)

Immunohistochemical staining of paraffin-embedded Human uterus tissue using ab108297 at a dilution of 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD99 antibody [EPR3096] (AB108297)

Immunohistochemical staining of paraffin-embedded Human tonsil tissue using ab108297 at a dilution of 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD99 antibody [EPR3096] (AB108297)

Fluorescent immunohistochemical analysis of paraffin-embedded human normal pancreas tissue using ab108297. Green-CD99 red-PI

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-CD99 antibody [EPR3096] (AB108297)

CD99 was immunoprecipitated from 0.35 mg HUVEC (Human umbilical vein endothelial cell) whole cell lysate 10 μg with 108297 at 1/50 dilution (2μg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HUVEC (Human umbilical vein endothelial cell) whole cell lysate 10 μg

Lane 2 : ab108297 IP in HUVEC whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab108297 in HUVEC whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

CD99 homodimer was reported in literature. (PMID : 18160845)

All lanes:

Immunoprecipitation - Anti-CD99 antibody [EPR3096] (ab108297)

Predicted band size: 19 kDa

Observed band size: 28 kDa,32 kDa

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• WB

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Western blot - Anti-CD99 antibody [EPR3096] (AB108297)

All lanes:

Western blot - Anti-CD99 antibody [EPR3096] (ab108297) at 1/1000 dilution

Lane 1:

THP-1 cell lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Lane 3:

U937 cell lysate at 10 µg

Lane 4:

Molt-4 cell lysate at 10 µg

Lane 5:

HuT-78 cell lysate at 10 µg

Lane 6:

HUVEC cell lysate at 10 µg

Predicted band size: 19 kDa

Observed band size: 28 kDa,32 kDa

false

• WB

CiteAb

Western blot - Anti-CD99 antibody [EPR3096] (AB108297)

CD99 western blot using anti-CD99 antibody [EPR3096] ab108297. Publication image and figure legend from Weekes, M. P., Tomasec, P., et al., 2014, Cell, PubMed 24906157.

ab108297 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab108297 please see the product overview.

Validation of Temporal Profiles by Flow Cytometry and Immunoblot and Investigation of Novel Immune Ligands, Related to Figure 4(A) PM and WCL profiles of MHC-I (HLA-A11) and CD99, validated by flow cytometry and immunoblot. ≥ 95% of HFFF were routinely infected by HCMV, assessed by flow cytometry for cell surface MHC-I.(B) Representative intracellular flow cytometry of 24h-infected HFFF with anti-IE1 confirms > 95% infection efficiency.(C) Flow cytometry of HFFF infected with HCMV confirms proteomic profiles for five additional cell surface proteins.(D and E) NK degranulation assays suggest that CLEC1A and FAT1 are novel activating NK ligands. Top panels – validation of temporal PM and WCL profiles by flow cytometry and immunoblot. CLEC1A was not quantified in any WCL QTV experiments but accumulated by immunoblot of whole-cell lysates, while depleting from the PM. Bottom panels – target cells underwent siRNA knockdown of CLEC1A (D) or FAT1 (E) and were then incubated with stimulated polyclonal NK cells from each of three donors. Degranulation of NK cells in response to both CLEC1A and FAT1 knockdown targets was significantly reduced compared to control. Error bars : +/- SEM (donors A, B), +/- range (donor C). * two-tailed p-value < 0.05, ** two-tailed p-value < 0.005. Cell surface MHC-I was unaffected by siRNA (right bottom panels, staining with W6-32 antibody or control Ig).(F) CD8+ T cell degranulation assay suggests that CEACAM1 is a novel inhibitory ligand for CMV-specific cytotoxic T cells. Top panel – validation of temporal PM profile by flow cytometry. Bottom panels (left) – flow cytometry of a CD8+ T cell line specific to the HCMV HLA-A2 restricted IE1 peptide VLEETSVML confirmed CEACAM1 surface expression. (right) HCMV peptide-specific CD8+ effector cells were incubated with autologous fibroblasts that had been infected with HCMV for 72h then pulsed with peptide or left unpulsed. Effectors and targets were treated with control Ig or anti-CEACAM1. HCMV-specific T cell degranulation was significantly increased with CEACAM1 block. Error bars ± SEM. * two-tailed p-value < 0.0001.y-axes of QTV plots represent relative abundance, and y-axes of flow cytometry plots represent % of max.

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