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Af1086

Manufactured by R&D Systems
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AF1086 is a high-quality lab equipment product designed for use in research and scientific applications. It is a specialized tool that serves a core function, but its intended use should not be extrapolated beyond the factual information provided.

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Lab products found in correlation

Erms da470, by Merck Group (2 mentions) Cc050, by Merck Group (2 mentions) Af164, by R&D Systems (2 mentions) Recombinant human gm csf, by Gentaur (2 mentions) Maxisorp plate, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Af156, by R&D Systems (1 mentions) Mab1086, by R&D Systems (1 mentions) Anti bcl6, by Santa Cruz Biotechnology (1 mentions) Anti pd 1, by R&D Systems (1 mentions) Sc 858, by Santa Cruz Biotechnology (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by Merck Group (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions) Mouse antihuman cd20 clone l26, by Agilent Technologies (1 mentions) Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions) Sp8 confocal microscope, by Leica (1 mentions)

8 protocols using af1086

1

Quantification of Soluble Immune Checkpoint Molecules

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Soluble co-signaling molecules were measured in serum samples by the ELISA technique. ELISA kits for soluble CD28 (BMS290), CD30 (BMS240MST), CD40 (BMS265MST), CD40L (BMS239MST), CD80 (BMS291INST), CD137 (BMS289), CTLA-4 (BMS276MST), and FAS (BMS245MST) were obtained from Bender MedSystems, Vienna, Austria, and used according to the manufacturer's instructions.
For soluble PD-1 and PD-L1 (B7-H1), microtiter plates were coated overnight at 4°C with 1 µg/ml of anti-human PD-1 antibody (AF1086, R&D Systems, UK) or anti-human B7-H1 antibody (AF156, R&D Systems). After blocking, serum samples were diluted 1∶4 for sPD-1 and 1∶2 for sPD-L1 molecule detection and purified recombinant human PD-17Fc (1086-PD, R&D Systems) and B7-H17Fc (156-B7, R&D Systems) chimeras were used as standards. Further, plates were incubated with 1 µg/ml of anti-human PD-1 monoclonal antibody (mAb, MAB1086, R&D Systems) or B7-H1 mAb (MAB156, R&D Systems), washed and a solution of conjugated goat anti-mouse IgG was added. Color reactions were developed using the TMB peroxidase substrate system (Sigma-Aldrich, St. Louis, MO) and measured at 450 nm. All samples were analyzed in duplicate and standard curves were run in each plate. No cross-reactivity between PD-1 and PD-L1 molecules was observed and intra- and inter-assay variability was evaluated to be <9% in all cases.
Melendreras S.G., Martínez-Camblor P., Menéndez A., Bravo-Mendoza C., González-Vidal A., Coto E., Díaz-Corte C., Ruiz-Ortega M., López-Larrea C, & Suárez-Álvarez B. (2014). Soluble Co-Signaling Molecules Predict Long-Term Graft Outcome in Kidney-Transplanted Patients. PLoS ONE, 9(12), e113396.
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2

Quantitative Antibody Binding ELISA

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Total IgGs were quantified using 96-well MaxiSorp plates (Nunc) coated with goat
anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470
(ERMs-DA470, Sigma-Aldrich) as a standard. To test specific binding of antibody
constructs, ELISA plates were coated with 2 μg ml−1 of type I
recombinant human collagen (Millipore, CC050), 2 μg ml−1 of an
anti-human LAIR1 antibody (clone DX26, BD Biosciences 550810), 1 μg
ml−1 of recombinant human GM-CSF (Gentaur), 2 μg
ml−1 of an anti-PD1 or an anti-SLAM antibody (R&D Systems,
AF1086 and AF164). Plates were blocked with 1% bovine serum albumin (BSA) and incubated
with titrated antibodies, followed by AP-conjugated goat anti-human IgG, Fcγ
fragment specific (Jackson Immuno Research, 109-056-098). Plates were then washed,
substrate (p-NPP, Sigma) was added and plates were read at 405 nm.
Pieper K., Tan J., Piccoli L., Foglierini M., Barbieri S., Chen Y., Silacci-Fregni C., Wolf T., Jarrossay D., Anderle M., Abdi A., Ndungu F.M., Doumbo O.K., Traore B., Tran T.M., Jongo S., Zenklusen I., Crompton P.D., Daubenberger C., Bull P.C., Sallusto F, & Lanzavecchia A. (2017). Public antibodies to malaria antigens generated by two LAIR1 insertion modalities. Nature, 548(7669), 597-601.
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3

Quantitative Antibody Binding ELISA

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Total IgGs were quantified using 96-well MaxiSorp plates (Nunc) coated with goat
anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470
(ERMs-DA470, Sigma-Aldrich) as a standard. To test specific binding of antibody
constructs, ELISA plates were coated with 2 μg ml−1 of type I
recombinant human collagen (Millipore, CC050), 2 μg ml−1 of an
anti-human LAIR1 antibody (clone DX26, BD Biosciences 550810), 1 μg
ml−1 of recombinant human GM-CSF (Gentaur), 2 μg
ml−1 of an anti-PD1 or an anti-SLAM antibody (R&D Systems,
AF1086 and AF164). Plates were blocked with 1% bovine serum albumin (BSA) and incubated
with titrated antibodies, followed by AP-conjugated goat anti-human IgG, Fcγ
fragment specific (Jackson Immuno Research, 109-056-098). Plates were then washed,
substrate (p-NPP, Sigma) was added and plates were read at 405 nm.
Pieper K., Tan J., Piccoli L., Foglierini M., Barbieri S., Chen Y., Silacci-Fregni C., Wolf T., Jarrossay D., Anderle M., Abdi A., Ndungu F.M., Doumbo O.K., Traore B., Tran T.M., Jongo S., Zenklusen I., Crompton P.D., Daubenberger C., Bull P.C., Sallusto F, & Lanzavecchia A. (2017). Public antibodies to malaria antigens generated by two LAIR1 insertion modalities. Nature, 548(7669), 597-601.
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4

Immunohistochemical Identification of Tfh Cells

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Immunohistochemistry was performed on formalin-fixed and paraffin-embedded 4-μM tissue sections. Sections were incubated with optimal concentrations of primary monoclonal antibodies, including anti-CD38, anti-CD20, anti-CD4, anti-PD-1 (R&D, AF1086, Abington, UK) and anti-Bcl-6 (Santa Cruz biotechnology, sc-858, Santa Cruz, CA, USA) for 1 hr at RT and then incubated with biotinylated goat anti-rat, goat-rabbit and biotinylated rabbit anti-mouse antibody (Zhongshan Goldenbridge Biotech, Beijing, China), respectively. The avidin-biotin-peroxidase system with 3-amino-9-ethyl-carbazole (AEC) (red color) or Vector blue (blue color) as substrates was used to perform double staining. Based on the double immunostaining, PD-1 and Bcl-6 double positive cells in liver tissue, and CXCR5 and CD4 double positive cells in the spleen were identified as Tfh cells. The absolute number of Tfh cells was independently counted by two pathologists from five representative high-power microscopic fields (400 magnification) relative to the area most involved by the inflammatory infiltrate (28 (link)).
Wang L., Sun Y., Zhang Z., Jia Y., Zou Z., Ding J., Li Y., Xu X., Jin L., Yang T., Li Z., Sun Y., Zhang J.Y., Lv S., Chen L., Li B., Gershwin M.E, & Wang F.S. (2015). CXCR5+ CD4+ T Follicular Helper Cells Participate in the Pathogenesis of Primary Biliary Cirrhosis. Hepatology (Baltimore, Md.), 61(2), 627-638.
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5

Immunoblotting Analysis of Protein Expression

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The immunoblotting study was performed using SDS-PAGE with 10% or 12% gel, and 15
µg total protein/lane was used for each cell lysate. After transferring the
proteins to polyvinylidene difluoride membranes, the membranes were hybridized
with antibodies against PD-1 (AF1086, R & D System), RANTES (CCL5; C-12,
Santa Cruz Biotechnology, Dallas, TX), CD19 (PDR134, Santa Cruz Biotechnology),
AKT1 (B1, Santa Cruz Biotechnology), or β-actin (C4, Santa Cruz Biotechnology).
Quantification of the proteins bands on X-ray films was done using an ImageJ
software (https://imagej.nih.gov/ij/).
Wang G., Wang L., Zhou J, & Xu X. (2019). The Possible Role of PD-1 Protein in Ganoderma lucidum–Mediated Immunomodulation and Cancer Treatment. Integrative Cancer Therapies, 18, 1534735419880275.
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6

Modulation of PBMC Immune Responses

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Cryopreserved PBMCs were defrosted, counted and used for flow cytometry directly or after overnight culture (37°C, 5% CO2) in round-bottom 96-well plates (300 000/200 µL/well) in RPMI 1640 medium, 10% pooled heat-inactivated human AB serum (Sigma-Aldrich), 100 IU/mL penicillin/streptomycin, 2.2 mmol/L l-Glutamine, 23 mmol/L HEPES (all from Gibco/Thermo Fisher Scientific). Cell recovery and viability were always greater than 90%. Cells were unstimulated or stimulated overnight with fixed E. coli (100 bacteria per cell (BpC)). One hour before adding E. coli, some cultures were (1) preblocked with polyclonal blocking antibody anti-PD1 (10 µg/mL, AF1086, BioTechne/R&D Systems, Abingdon, UK); (2) preincubated with ARC/SAH plasma (10%); (3) pretreated with increasing concentrations of ethanol (50–100–250 mmol/L); or (4) preincubated with ARC/SAH/HC faecal extracts (FEB, 100 BpC). Brefeldin A (Sigma-Aldrich) was added to cultures used for intracellular flow cytometry 1 hour after E. coli (10 µg/mL).
Riva A., Patel V., Kurioka A., Jeffery H.C., Wright G., Tarff S., Shawcross D., Ryan J.M., Evans A., Azarian S., Bajaj J.S., Fagan A., Patel V., Mehta K., Lopez C., Simonova M., Katzarov K., Hadzhiolova T., Pavlova S., Wendon J.A., Oo Y.H., Klenerman P., Williams R, & Chokshi S. (2017). Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease. Gut, 67(5), 918-930.
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7

Modulation of Chemotherapy Response by PD-1 Signaling

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MDA-MB-231, DU145 and 4T1 cells were incubated with recombinant PD-1 (rPD-1; R&D Systems; human: #1086-PD; mouse: #1021-PD) in serum-free medium for 24 h prior to exposure to chemotherapeutic agents and subsequent clonogenic assays. Alternatively, MDA-MB-231 or DU145 cells were co-cultured with human Jurkat T cells for 24 h in a 1:5 ratio. Jurkat cells were stimulated with IL-2 (100U; Sigma, #17908-10KU) prior to co-culture in order to induce activation and stimulate PD-1 expression as previously reported [26 (link)]. We have shown that > 90% of Jurkat T cells express PD-1 [13 (link)]. In some experiments, we blocked PD-1/PD-L1 interaction with either human anti-PD-L1 antibody (4 μg/ml; Biolegend #329702) or anti-PD-1 antibody (1 μg/ml; R&D Systems #AF1086) prior to exposure to rPD-1 or Jurkat T cells and doxorubicin.
Black M., Barsoum I.B., Truesdell P., Cotechini T., Macdonald-Goodfellow S.K., Petroff M., Siemens D.R., Koti M., Craig A.W, & Graham C.H. (2016). Activation of the PD-1/PD-L1 immune checkpoint confers tumor cell chemoresistance associated with increased metastasis. Oncotarget, 7(9), 10557-10567.
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8

Multiplex Immunofluorescence Staining of Tissue Sections

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Immunofluorescence staining was performed on serial sections for CD20, Ki67, CD3, PD1, CXCR5 and DAPI dye, using a modified protocol previously described (33 (link)). Briefly, 4 μm paraffin-embedded tissue sections were subjected to deparaffinization in xylene, rehydration in graded series of ethanol, and rinsing with distilled water. Heat-mediated epitope retrieval was performed with DIVA decloaker, followed by blocking with 10% normal donkey serum (Jackson ImmunoResearch) for 1 hour. Sections were incubated with optimized concentrations of rabbit antihuman-Ki67 (clone SP6, Abcam), mouse antihuman CD20 (clone L26, Dako), Rat anti Human CD3 (clone CD3-12, BIO-RAD), goat polyclonal antihuman PD-1 (AF1086, R& D systems) and rabbit polyclonal anti CXCR5 (HPA042432, Sigma) overnight. Thereafter, the sections were washed and incubated with conjugated secondary Abs (Alexa Fluor 488/568/647, Abcam) at room temperature for 1 hour. Following incubations, the slides were washed twice with PBS-FSG-Tx100 for ten minutes. Upon completion of immunofluorescence staining, the sections were mounted with ProLong® Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) (Life Technologies) as a nuclear counterstain and coverslipped. Images were captured using a Leica SP8 confocal microscope.
Silveira E.L., Rogers K.A., Gumber S., Amancha P., Xiao P., Woollard S.M., Byrareddy S.N., Teixeira M.M, & Villinger F. (2017). Immune cell dynamics in rhesus macaques infected with a Brazilian strain of Zika virus. Journal of immunology (Baltimore, Md. : 1950), 199(3), 1003-1011.
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