Pd l1

Manufactured by Cell Signaling Technology

Sourced in United States, China, United Kingdom

PD-L1 is a protein that plays a role in the regulation of the immune system. It is expressed on the surface of various cell types and interacts with the PD-1 receptor, which is present on T cells. This interaction can inhibit T cell activation and proliferation, potentially affecting the immune response. PD-L1 is involved in the process of immune checkpoint regulation.

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184 protocols using pd l1

Comprehensive Immunodetection Protocol

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For western blotting immunodetection the following antibodies were used: NF-κB (1:1000, Santa Crus, SC-8008), PD-L1 (1:1000, Cell Signaling, #13684 S), CD63 (1:1000, R&D Systems, MAB50482), CD9 (1:1000, Cell Signaling Technology, 13403 S), Flotillin-1 (1:1000, Abcam, ab133497), and Actin (1:1000, Licor, 926–42212).
For immunocytochemistry and immunohistochemistry applications the following antibodies were used: Desmin (1:250, Abcam, ab32362), NF-κB (1:200, Santa Crus, SC-8008), PD-L1 (1:200, Cell Signaling, #13684 S), Mφ (1:100, LifeSpan BioScience, LS-B9966), CD163 (1:200, Abcam, ab87099), Pax7 (1:100, ThermoFisher, PA1-117), MyoD (5.8 A) (1:200, ThermoFisher, MA1-41017), and Heavy Chain Cardiac Myosin (1:100, Abcam, ab50967). A complete table of antibodies can be found in Supplemental Table 10.

Rolland T.J., Peterson T.E., Singh R.D., Rizzo S.A., Boroumand S., Shi A., Witt T.A., Nagel M., Kisby C.K., Park S., Rowe L.A., Paradise C.R., Becher L.R., Paradise B.D., Stalboerger P.G., Trabuco E.C, & Behfar A. (2022). Exosome biopotentiated hydrogel restores damaged skeletal muscle in a porcine model of stress urinary incontinence. NPJ Regenerative Medicine, 7, 58.

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Comprehensive Molecular Profiling Techniques

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Western blotting: TIMELESS (Abcam, ab109512, 1/50000), β-actin (Abcam, ab49900, HRP-linked,1/50000), c-Myc (Abcam, ab32072, 1/1000) and PD-L1 (Cell Signaling Technology, #13684, 1/1000), Rabbit secondary antibody (Cell Signaling Technology, #7074, 1/2000).
Immunohistochemistry: TIMELESS (Abcam, ab72458, 1/200), CD8a (Servicebio, GB13429, 1/200), PD-L1 (Servicebio, GB11339A, 1/500), Ki-67 (Servicebio, GB111141, 1/500), Mouse secondary antibody (Servicebio, GB23301, 1/200), Rabbit secondary antibody (Servicebio, GB23303, 1/200).
Immunofluorescence: TIMELESS (Abcam, ab109512, 1/100), c-Myc (Abcam, ab32072, 1/100), CD8a (Servicebio, GB13429, 1/5000), Granzyme B (Abcam, ab255598, 1/2000), active caspase 3 (Servicebio, GB11532, 1/500), Hoechst 33342 (Beyotime, C1027, 1/100), CY3-TSA (Apex Bio, K1051, 1/200), Donkey Anti-Rabbit IgG H&L Alexa Fluor^® 647 (Abcam, ab150075, 1/200).
Flow cytometry: PD-L1 (Cell Signaling Technology, #13684, 1/200), CD8a-PerCP/Cyanine5.5 (Clone 53-6.7, Biolegend 1007341/100), CD45-AF700 (Biolegend 103128, 1/80), Fixable Viability Dye eFluor™ 450 (Invitrogen, 1/500).

Dong X., Dai H., Lin Y., Sheng X., Li Y., Wang Y., Zhang X., Jiang S., Yin W, & Lu J. (2023). TIMELESS upregulates PD-L1 expression and exerts an immunosuppressive role in breast cancer. Journal of Translational Medicine, 21, 400.

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Western Blot Analysis of PD-L1 and LMP1

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The cells and medium were collected 48 h after seeding the cells in 10 cm dishes. The total cell lysates were denatured in radioimmunoprecipitation assay lysis buffer (Tris-HCI, 1% NaCl, sodium deoxycholate, sodium dodecyl surface [SDS], sodium orthovanadate, NaF, and fluoride) and boiled for 5 min. The medium was concentrated to 50 fold using Vivaspin VS2041 (Sartorius, Goettingen, Germany). The samples were separated using SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% skim milk Tris-buffered saline-Tween 20 (TBST) and incubated at 4 °C overnight with the following primary antibodies: PD-L1 (#13684, 1:1000, Cell Signaling Technology, Danvers, MA), LMP1 (M0897, 1:200, Dako, Glostrup, Denmark), and α-tubulin (T9026, 1:5000, Sigma Aldrich, St. Louis, MO, USA). After washing with TBST (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the membranes were exposed to horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories, Hercules, CA, USA). The signals were detected using enhanced chemiluminescence reagent (GE Healthcare, Tokyo, Japan).

Kase K., Kondo S., Wakisaka N., Dochi H., Mizokami H., Kobayashi E., Kano M., Komori T., Hirai N., Ueno T., Nakanishi Y., Hatano M., Endo K., Moriyama-Kita M., Sugimoto H, & Yoshizaki T. (2021). Epstein–Barr Virus LMP1 Induces Soluble PD-L1 in Nasopharyngeal Carcinoma. Microorganisms, 9(3), 603.

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Gastric Tumor Protein Profiling

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The total protein of gastric tumor tissues and paired adjacent non-tumorous tissues was extracted using a mammalian protein extraction kit (Kangwei, China), and the concentration of various proteins was measured using a BCA kit (Kangwei, China). The levels of PD-1 (1:200, Abcam), PD-L1 (1:2000, Cell Signaling Technology), PD-L2 (1:500, R&D Systems), and GAPDH (1:1000, Abcam) were measured with ECL reagents (Thermo Fisher Scientific) using Molecular Imager Chemi Dox XRS+ imaging system (Biorad, California, USA).

Wu Y., Cao D., Qu L., Cao X., Jia Z., Zhao T., Wang Q, & Jiang J. (2017). PD-1 and PD-L1 co-expression predicts favorable prognosis in gastric cancer. Oncotarget, 8(38), 64066-64082.

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Immunohistochemical Analysis of Checkpoint Proteins

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Immunohistochemical (IHC) staining was performed on TMAs using the following primary antibodies: PD-L1 (1:200, 4.37 µg/mL, 13684, Cell Signaling Technology), B7-H3 (1:400, 1.49 µg/mL, 14 058S, Cell Signaling Technology), B7-H4 (1:400, 0.66 µg/mL, 14572, Cell Signaling Technology) and VISTA (1:200, 0.05 µg/mL, 54979, Cell Signaling Technology).15–18 (link) TMA slides were stained using standard immunohistochemistry techniques as previously described.15 (link) The IHC staining was scanned using Pannoramic MIDI (3D HISTECH). The histoscore of the membrane and nuclear staining quantification was assessed according to the formula (3+ per cent cells)×3+(2+per cent cells)×2+(1+per cent cells)×1, and the formula total intensity/total cell number was used to assess the histoscore of pixel quantification. In this case, the normalized score is between 0 and 300. This method has previously been described.19 (link)

Wang Y., Deng J., Wang L., Zhou T., Yang J., Tian Z., Yang J., Chen H., Tang X., Zhao S., Zhou L., Tong A, & Xu J. (2020). Expression and clinical significance of PD-L1, B7-H3, B7-H4 and VISTA in craniopharyngioma. Journal for Immunotherapy of Cancer, 8(2), e000406.

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Multiplex IHC for Immune Cell Profiling

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Two 4-µm sections from TMA blocks were subjected to mfIHC using the PANO Multiplex IHC kit (0004100100, Panovue, Beijing, China) to examine specific cell markers including CD11c (ab52632, Abcam), CD45RO (55618, Cell Signaling), CD68 (ZM0060, ZSGB-Bio), panCK (4545, Cell Signaling), and PD-L1 (13684, Cell Signaling) in panel A, and CD4 (BX50023, biolynx), CD8A (70306, Cell Signaling), CD56 (3576, Cell Signaling), FoxP3 (320202, Biolegend), and granzyme B (ab4059, Abcam) in panel B. Different primary antibodies were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody incubation and TSA. The slides were microwave heat-treated after each TSA operation. Nuclei were stained with 4ʹ-6ʹ-diamidino-2-phenylindole (DAPI, D9524, Sigma-Aldrich) after all the human antigens had been labeled.

Pan C., Wang Y., Liu Q., Hu Y., Fu J., Xie X., Zhang S., Xi M, & Wen J. (2021). Phenotypic profiling and prognostic significance of immune infiltrates in esophageal squamous cell carcinoma. Oncoimmunology, 10(1), 1883890.

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Molecular Profiling of DNA Repair Pathways

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PD-L1 (#13684), PARP1 (#9532), phospho-GSK3β (Ser9, #9336), GSK3β (#9315), Ki-67 (#9449) antibodies were purchased from Cell Signaling Technology, BRCA1 (sc-8326) and BRCA2 (sc-642) were from Santa Cruz, and α-Tubulin (#B-5-1-2) and β-Actin (#A2228) were obtained from Sigma-Aldrich (St. Louis, MO). Olaparib, rucaparib, and talazoparib were purchased from Selleckchem (Houston, TX), ChemieTek (Indianapolis, IN), and Selleckchem, respectively. Human CD274 (B7-H1, PD-L1) antibody for T-cell killing assay was from BioLegend (#329709). eSiRNA human BRCA1 (EHU096311), eSiRNA human BRCA2 (EHU031451) and siRNA universal negative control (SIC001) were from Sigma-Aldrich.

Jiao S., Xia W., Yamaguchi H., Wei Y., Chen M.K., Hsu J.M., Hsu J.L., Yu W.H., Du Y., Lee H.H., Li C.W., Chou C.K., Lim S.O., Chang S.S., Litton J., Arun B., Hortobagyi G.N, & Hung M.C. (2017). PARP inhibitor upregulates PD-L1 expression and enhances cancer-associated immunosuppression. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(14), 3711-3720.

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Lung Nodule Protein Profiling

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Lung nodules were homogenized, lysed using RIPA (Santa Cruz, TX), and 10% SDS/PAGE was run after protein quantification (Bio‐Rad, Hercules, CA). Primary antibodies (1:1000): PD‐L1, p‐Stat3‐705, Stat‐3, phospho‐p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, cleaved caspase‐3 (Asp175), and caspase‐3 (Cell Signaling, MA) were used. Autoradiography detection and quantification were performed using Image J.

Dhupkar P., Gordon N., Stewart J, & Kleinerman E.S. (2018). Anti‐PD‐1 therapy redirects macrophages from an M2 to an M1 phenotype inducing regression of OS lung metastases. Cancer Medicine, 7(6), 2654-2664.

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Protein Extraction and Antibody Immunoblotting

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Cells were harvested and proteins were extracted with RIPA lysis buffer. The following antibodies were used: NPM1 (FC-61991; Invitrogen, Carlsbad, CA, USA; 1:1000), PD-L1 (GTX104763; GeneTex, Irvine, CA, USA; 1:4000), PD-L1 (GTX31308; GeneTex; 1:1000), PD-L1 (13684; Cell Signaling Technology, Danvers, MA, USA; 1:1000), PARP1 (9532; Cell Signaling Technology; 1:1000), Flag (14793; Cell Signaling Technology; 1:1000), Myc (16286-1-AP; Proteintech, Rosemont, IL, USA; 1:1000), HSP70 (46477; Cell Signaling Technology; 1:1000), GAPDH (10494-1-AP; Proteintech; 1:10,000). Cytokine IFN-γ (EST-IFg-0100; Stemimmune LLC, Richmond, CA, USA) was used to treat cells at the concentration of 25 μg mL⁻¹.

Qin G., Wang X., Ye S., Li Y., Chen M., Wang S., Qin T., Zhang C., Li Y., Long Q., Hu H., Shi D., Li J., Zhang K., Zhai Q., Tang Y., Kang T., Lan P., Xie F., Lu J, & Deng W. (2020). NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer. Nature Communications, 11, 1669.

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Hematoxylin Staining and Immune Markers

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Hematoxylin staining and evaluation of the tissue architecture was used to define the stromal type. The tumors were stained with the following antibodies: PD-L1 (Cell Signaling, dilution 1:500), PTRH1 (BIOSS, dilution 1:200). Expression of PD-L1 was evaluated by counting specific cytoplasmic staining cells in 5 randomly selected areas at 40 × light for each group. The average immunity group image gradation analysis integral light density of PTRH1 in 5 randomly selected areas for each group were calculated by using Imagepro Plus 6.0 (Media Cybernetics, Inc).

Gao C., Chen J., Bai J., Zhang H., Tao Y., Wu S., Li H., Wu H., Shen Q, & Yin T. (2023). High glucose-upregulated PD-L1 expression through RAS signaling-driven downregulation of PTRH1 leads to suppression of T cell cytotoxic function in tumor environment. Journal of Translational Medicine, 21, 461.

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