CD279 (PD-1) Monoclonal Antibody (J43), PE, eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The cells were counted and 1×106 cells were used for surface staining. For intracellular staining 1×106 cells were cultured per well in 24 well plates (Nunc, USA) and activated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 750 ng/ml ionomycin (Sigma, USA) overnight, and 10 µg/ml brefeldin A (eBiosciences, USA) was added during the last 4 hours of culture. Cells were washed twice with PBS and stained with antibodies directed against surface markers	After staining, cells were washed again with PBS and cells were fixed with 100 µl fixation buffer (eBiociences, USA) for 30 minutes, then re-suspended in 200 µl permeabilization buffer (eBiosciences, USA) and stained with fluorescently labelled anti-cytokine antibodies. Fluorescence intensity of fluorochrome-labelled cells was measured by flow cytometry (FACS Canto™ II, BD Biosciences, USA).

Protocol tips
The cells were counted and 1×106 cells were used for surface staining. For intracellular staining 1×106 cells were cultured per well in 24 well plates (Nunc, USA) and activated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 750 ng/ml ionomycin (Sigma, USA) overnight, and 10 µg/ml brefeldin A (eBiosciences, USA) was added during the last 4 hours of culture. Cells were washed twice with PBS and stained with antibodies directed against surface markers
Downstream tips
After staining, cells were washed again with PBS and cells were fixed with 100 µl fixation buffer (eBiociences, USA) for 30 minutes, then re-suspended in 200 µl permeabilization buffer (eBiosciences, USA) and stained with fluorescently labelled anti-cytokine antibodies. Fluorescence intensity of fluorochrome-labelled cells was measured by flow cytometry (FACS Canto™ II, BD Biosciences, USA).

Publication protocol

Spleens were isolated from PD-1−/− and WT mice, either infected or uninfected, and macerated by frosted slides in 10% RPMI 1640 (Gibco, Invitrogen, UK) and made into a single cell suspension. Red blood cells (RBCs) were lysed with RBC cell lysis buffer, incubated at room temperature for three to five minutes and washed with 10% RPMI 1460. The cells were counted and 1×106 cells were used for surface staining. For intracellular staining 1×106 cells were cultured per well in 24 well plates (Nunc, USA) and activated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 750 ng/ml ionomycin (Sigma, USA) overnight, and 10 µg/ml brefeldin A (eBiosciences, USA) was added during the last 4 hours of culture. Cells were washed twice with PBS and stained with antibodies directed against surface markers. After staining, cells were washed again with PBS and cells were fixed with 100 µl fixation buffer (eBiociences, USA) for 30 minutes, then re-suspended in 200 µl permeabilization buffer (eBiosciences, USA) and stained with fluorescently labelled anti-cytokine antibodies. Fluorescence intensity of fluorochrome-labelled cells was measured by flow cytometry (FACS Canto™ II, BD Biosciences, USA). LC3-B staining was performed according to the manufacturer's protocol (Cell Signalling, USA) and cell viability dye (7-AAD) was added to the LC3-B stained cells 15 minutes before analyzing the cells by flow cytometry. FACS Diva was used for acquiring the cells and final data analysis was performed by Flow Jo (Tree star, USA).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Flow cytometry Anti-bodies Mouse - CD279/PD-1 using CD279 (PD-1) Monoclonal Antibody (J43), PE, eBioscience™ from eBioscience.

Manufacturer protocol

Download the product protocol from eBioscience for CD279 (PD-1) Monoclonal Antibody (J43), PE, eBioscience™ below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Flow cytometry Anti-bodies Mouse - CD279/PD-1 using CD279 (PD-1) Monoclonal Antibody (J43), PE, eBioscience™ from eBioscience. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.