Immunofluorescence analysis of CD28 Monoclonal Antibody (10F3) was performed using 70% confluent log phase Jurkat cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with CD28 (10F3) Mouse Monoclonal Antibody (CD28004) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membranous localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human|
|Host / Isotype||Mouse / IgG1|
|Storage buffer||PBS with BSA|
|Contains||0.1% sodium azide|
|Storage Conditions||4° C, store in dark|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:20|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
CD28 antigen is a 44 kDa disulfide linked homodimeric T cell specific surface glycoprotein. It is a cell adhesion molecule of the immunoglobulin superfamily which is constitutively expressed on most peripheral blood T lymphocytes (approximately 95% of CD4+ cells and 50% of CD8+ cells). Mature thymocytes exhibit higher levels of CD28 than the immature cells. Activation of T cells results in enhanced CD28 expression. T cell activation requires two combined signals provided by antigen presenting cells.
Analyte Specific Reagent