Recombinant Anti-PD1 antibody [NAT105] (ab234444)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab234444 (right) or Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control (left). PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab234444 or Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control (1x 10⁶ in 100 µl at 5.0 μg/ml (1/516)) for 30min on ice. The cells were simultaneously stained with CD3.

The secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on viable cells.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling PD1 with ab234444 at 1/50 dilution, followed by Rabbit Anti-Mouse IgG + Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining on T cells of human tonsil germinal center is observed. Performed on a Leica Biosystems BOND instrument. Counter stained with hematoxylin.

Ab234444 staining PD1 in the MOLT-4 (human lymphoblastic leukemia T lymphoblast) treated with Ionomycin (500 ng/ml, 24 h) and PMA (10 ng/ml, 24 h) by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde, permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/50). ab150117 anti-mouse IgG H&L (Alexa Fluor® 488) preadsorbed (1/100) was used as the secondary antibody. 

ab179504 Anti-beta IV Tubulin (1/200) was used as a counter stain, detected by ab150080 AlexaFluor®594 Goat anti- Rabbit secondary (1/500)

DAPI was used as a Nuclear counter stain.

Intracellular flow cytometric analysis ofMOLT-4 (human lymphoblastic leukemia cell line) cell line treated with ionomycin (500 ng/ml, 24h) and PMA (10 ng/ml, 24h)labeling PD1 with ab234444 at 1/1000 dilution (red) and an untreated control (green) compared with aMouse monoclonalIgG1 (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (ab150113) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells. 

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID: 26318293)

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