Pd l1 clone 22c3

Manufactured by Agilent Technologies

Sourced in United States, Denmark, China

The PD-L1 (clone 22C3) is a lab equipment product offered by Agilent Technologies. It is a monoclonal antibody designed for the detection of the programmed death-ligand 1 (PD-L1) protein in biological samples. The core function of this product is to enable the identification and quantification of PD-L1 expression levels in cells or tissues.

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Lab products found in correlation

Cd8 clone c8 144b, by Agilent Technologies (3 mentions) Ez prep solution, by Roche (3 mentions) Cell conditioning 1 cc1, by Roche (3 mentions) Optiview dab detection kit, by Roche (3 mentions) Hematoxylin and bluing reagent, by Roche (2 mentions) Benchmark ultra, by Roche (2 mentions) Envision dual link system hrp dab, by Agilent Technologies (1 mentions) Ventana benchmark ultra, by Roche (1 mentions) Dako envision flex, by Agilent Technologies (1 mentions) Leica rm2255 automated microtome, by Leica Biosystems (1 mentions) Dako omnis immunostainer, by Agilent Technologies (1 mentions) Lenvatinib, by Selleck Chemicals (1 mentions) Matrigel, by BD (1 mentions) Anti hif1a, by Novus Biologicals (1 mentions) Benchmark automated immunostainer, by Roche (1 mentions) Benchmark, by Roche (1 mentions) Hematoxylin 2 and bluing reagent, by Roche (1 mentions) Benchmark ultra autostainer, by Roche (1 mentions) Optiview universal dab detection kit, by Roche (1 mentions) Ventana benchmark ultra platform, by Roche (1 mentions)

25 protocols using pd l1 clone 22c3

Characterization of Patient-Derived CAFs

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We characterized patient-derived CAFs by testing the expression of SMA (Actin, Smooth Muscle, 1A4; Cell Marque catalog # 202M-94), S100A4 (Recombinant Anti-S100A4 antibody; Abcam catalog # ab124805), TE-7 (Fibroblasts Antibody, TE-7; NOVUS Biologicals, LLC, Centennial, CO, USA; catalog # NBP2-50082), and negative expression of epithelial tumor markers including EpCAM (Ep-CAM/Epithelial Specific Antigen, Ber-EP4; Cell Marque, Rocklin, CA, USA; catalog # 248M-94) as well as CK 8, 18 (Cytokeratin 8 and 18; B22.1 and B23.1 from Cell Marque; catalog # 818M-94). Human uterine fibroblasts (HUF) and NCI-H441 tumor cell lines were used for validation. The percentage of PD-L1 (PD-L1 [Clone 22C3]; Agilent-Dako, Santa Clara, CA, USA; catalog # M365329-1) expression was tested in well-characterized CAFs from both tumors and tumor-adjacent normal samples. The IHC detection kit was procured from Dako (Envision+ Dual-link system-HRP (DAB+), code K4065).

Sulaiman R., De P., Aske J.C., Lin X., Dale A., Koirala N., Gaster K., Espaillat L.R., Starks D, & Dey N. (2023). Tumor-TME Bipartite Landscape of PD-1/PD-L1 in Endometrial Cancers. International Journal of Molecular Sciences, 24(13), 11079.

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Evaluating Tumor Characteristics and Immune Markers in Squamous Cell Carcinoma

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We evaluated tumor budding, nuclear size, stromal content, and spread through alveolar spaces using representative slides of SCCs. Tumor budding was assessed in areas showing the most extensive budding activity. Tumor budding was defined as a tumor nest composed of less than five tumor cells. Tumor stromal content was categorized as low (<50% of the entire tumor area) and high (≥50%). The presence of spread through alveolar spaces was defined as tumor cell nests or single cells spreading within air spaces beyond the edge of the main tumor. Immunohistochemical examination was performed on TMAs of SCCs using antibodies against p40 (BC28; Biocare, Copenhagen, Denmark) and programmed death‐ligand 1 (PD‐L1; clone 22C3; DAKO/Agilent Technologies, Glostrup, Denmark), following the manufacturers' recommendations. Tumors were considered PD‐L1 positive if ≥1% of the tumor cells were stained, i.e. tumor proportion score ≥1%. In situ hybridization for Epstein–Barr virus‐encoded RNA (Roche, Tucson, AZ, USA) was performed on TMAs of SCCs, following the manufacturer's recommendations.

Ura A., Hayashi T., Komura K., Hosoya M., Takamochi K., Sato E., Saito S., Wakai S., Handa T., Saito T., Kato S., Suzuki K, & Yao T. (2023). Copy number loss of KDM5D may be a predictive biomarker for ATR inhibitor treatment in male patients with pulmonary squamous cell carcinoma. The Journal of Pathology: Clinical Research, 10(1), e350.

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Immunohistochemical Analysis of Tumor-Infiltrating CD8+ T Cells and PD-L1 Expression

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The biopsy specimens were embedded in paraffin, and immunohistochemical (IHC) staining was performed on 5-μm-thick tissue sections using affinity mouse monoclonal antibodies against CD8 (clone C8-144B, dilution 1:1; Maixin Biotech, Fuzhou, China) and PD-L1 (clone 22c3, dilution 1:40, Agilent Tech, Inc., USA). Tissue sections were deparaffinized and stained using the Ventana BenchMark ULTRA in automatic mode (Roche Diagnostics GmbH, Germany) for analyzing CD8+ T-cell infiltration and PD-L1 expression in tumor cells.

Zhou P., Chen D., Zhu B., Chen W., Xie Q., Wang Y., Tan Q., Yuan B., Zuo X., Huang C., Zhu H, & Li G. (2021). Stereotactic Body Radiotherapy Is Effective in Modifying the Tumor Genome and Tumor Immune Microenvironment in Non-Small Cell Lung Cancer or Lung Metastatic Carcinoma. Frontiers in Immunology, 11, 594212.

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Immunohistochemical Detection of PD-L1 in Tissue Samples

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Slides were stained for routine diagnostics, over a period of several years. Formalin‐fixed paraffin‐embedded blocks were cut into 3‐µm sections with a Leica RM2255 Automated Microtome (Leica Biosystems B.V., Amsterdam, the Netherlands). Sections were placed on microscope slides and dried at either 60°C for 30 min to 16 h, or at 37°C for 72 h. After being dried, the slides were deparaffinised, and antigen retrieval was performed in citrate buffer (Target Retrieval Solution, pH 6) for 40 min. Immunohistochemistry (IHC) was performed according to a laboratory‐developed test protocol. Slides were stained with the Dako Omnis immunostainer and Dako EnVision Flex+ reagents and 1:20 dilution of PD‐L1 clone 22C3 (Dako Omnis, Dako Agilent Technologies, Leuven, Belgium). The IHC slides were then counterstained with haematoxylin, and coverslips were applied. Tonsil and placental tissue were used as positive controls for PD‐L1 expression.

Hondelink L.M., Hüyük M., Postmus P.E., Smit V.T., Blom S., von der Thüsen J.H, & Cohen D. (2021). Development and validation of a supervised deep learning algorithm for automated whole‐slide programmed death‐ligand 1 tumour proportion score assessment in non‐small cell lung cancer. Histopathology, 80(4), 635-647.

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Characterizing Uterine Fibroblasts and HUVEC Cells

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Human uterine fibroblasts (HUF; Primary Uterine Fibroblasts, Cat # PCS-460-010) and HUVEC cells were procured from ATCC (cat # PCS-100-013) and were cultured according to the standard cell culture procedures as per ATCC. Other cell lines for qRT-PCR were procured from ATCC. Matrigel was procured from BD biosciences, USA. Fluorescence vital stains, DiI, and DiO were bought from Molecular Probes, USA. Lenvatinib was procured from Selleckchem, PA, USA. Fibroblasts Antibody (TE-7) and recombinant Anti-S100A4 antibodies were procured from NOVUS and Abcam, respectively. PD-L1 [Clone 22C3] was bought from Agilent-Dako. PD-L2 (D7U8C) and CD31 were procured from Cell Signaling, USA. Cytokeratin 8 and 18 (B22.1 and B23.1), Ep-CAM/Epithelial Specific Antigen (Ber-EP4), Actin, Smooth Muscle (1A4), and Vimentin (SP20) were bought from Cell Marque, USA. Primers for qRT-PCR (Primer pairs are ordered from Integrated DNA Technologies, Inc. [IDT], Iowa, USA) of primary CAF are presented below:

Sulaiman R., De P., Aske J.C., Lin X., Dale A., Koirala N., Gaster K., Espaillat L.R., Starks D, & Dey N. (2023). Patient-Derived Primary Cancer-Associated Fibroblasts Mediate Resistance to Anti-Angiogenic Drug in Ovarian Cancers. Biomedicines, 11(1), 112.

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Multiplex Immunostaining of TFE3, HIF1A, CD31, CD8, and PD-L1

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IHC and multiple immunofluorescence were performed as previously described^{32 (link)}. Commercially available primary anti-TFE3 (clone MRQ-37, 1:100, MXB biotechnologies, Fujian, China), anti-HIF1A (1:5000, Novus Biologicals, Colorado, USA), anti-CD31 (clone UMAB30, ready to use, ZSGB-BIO, Beijing, ChinaMXB), CD8 (clone C8/144B, ready to use, Dako, Copenhagen, DEN) and PD-L1 (clone 22C3, 1:50, Dako) were used in this study. Multiplex immunofluorescence staining was performed with a PANO 7-plex kit (0004100100, Panovue, Beijing, CHN). CD8, macrophage, and NK cells expression were quantified as positive cell density (cell number per mm²). PD-L1 expression was assessed by tumor proportion score, which was defined as the percentage of tumor cells with membranous PD-L1 staining. PD-L1 expression >1% was defined as positivity.

Sun G., Chen J., Liang J., Yin X., Zhang M., Yao J., He N., Armstrong C.M., Zheng L., Zhang X., Zhu S., Sun X., Yang X., Zhao W., Liao B., Pan X., Nie L., Yang L., Chen Y., Zhao J., Zhang H., Dai J., Shen Y., Liu J., Huang R., Liu J., Wang Z., Ni Y., Wei Q., Li X., Zhou Q., Huang H., Liu Z., Shen P., Chen N, & Zeng H. (2021). Integrated exome and RNA sequencing of TFE3-translocation renal cell carcinoma. Nature Communications, 12, 5262.

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PD-L1 Expression Assessment by IHC 22C3 Assay

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The PD‐L1 IHC 22C3 pharmDx assay²⁶, ²⁷ was used to assess PD‐L1 expression in formalin‐fixed tumour samples obtained at the time of diagnosis of metastatic disease. PD‐L1 clone 22C3 is from Dako and stained on Roche Ventana Benchmark Ultra ISH/IHC autostainer with Optiview Polymer Detection Kit. Interpretation of PD‐L1 expression is based on the interpretation guide provided by Dako and was characterised according to tumour proportion score (TPS).²⁸

Low J.L., Huang Y., Sooi K., Ang Y., Chan Z.Y., Spencer K., Jeyasekharan A.D., Sundar R., Goh B.C., Soo R, & Yong W.P. (2021). Low‐dose pembrolizumab in the treatment of advanced non‐small cell lung cancer. International Journal of Cancer, 149(1), 169-176.

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Immunohistochemical Analysis of PD-L1 and CMTM6

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Immunohistochemistry of the formalin-fixed paraffin-embedded samples was
performed on a BenchMark Ultra autostainer (Ventana Medical Systems). Briefly, 3
µm paraffin serial sections were cut, heated at 75°C for 28
minutes and deparaffinised in the instrument with EZ prep solution (Ventana
Medical Systems). Heat-induced antigen retrieval was carried out using Cell
Conditioning 1 (CC1, Ventana Medical Systems) for 48’ for PD-L1, and
64’ for CMTM6 antibodies at 95°C.
PD-L1 clone 22C3 (Dako) was used at 1:40 dilution, 1 hour at room
temperature and CMTM6 clone 1D6 was used directly from hybridoma supernatant at
either 1:500 or 1:1000 dilution for tumor samples and 1:100 dilution for cell
lines, 1 hour at room temperature. Bound antibody was detected using the
OptiView DAB Detection Kit (Ventana Medical Systems). Slides were counterstained
with Hematoxylin and Bluing Reagent (Ventana Medical Systems).
Patient melanoma samples were obtained (following Institutional Review
Board approval) from the NKI-AVL pathology archive biobank and selected for
PD-L1 expression.

Mezzadra R., Sun C., Jae L.T., Gomez-Eerland R., de Vries E., Wu W., Logtenberg M.E., Slagter M., Rozeman E.A., Hofland I., Broeks A., Horlings H.M., Wessels L.F., Blank C.U., Xiao Y., Heck A.J., Borst J., Brummelkamp T.R, & Schumacher T.N. (2017). Identification of CMTM6 and CMTM4 as PD-L1 protein regulators. Nature, 549(7670), 106-110.

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Quantifying Tumor Immune Profiles

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Four-micrometer-thick tumor sections were stained for PD-L1 (clone 22C3, DAKO, Agilent, CA, USA), CD4 (clone BP6028; Biolynx, Hangzhou, China), CD8 (clone: EP334, Abcam, Cambrige, UK) using BenchMark automated immunostainer (Ventana, AZ, USA). Staining was evaluated in a blinded fashion by the pathologists. Scoring was assessed based on the proportion of positive cells among nucleated cells in the ROI. The positive cells were defined as tumor cells displaying membranous staining of PD-L1. The PD-L1 expression at MPC was quantified as the proportion of PD-L1-positive tumor cells in total tumor cells within the MPC area. The tumor proportion score (TPS) is a PD-L1 measurement that has been applied in clinical trials and in clinic of lung cancer (20 (link)). The percentage of CD4+ and CD8+ T cell in the peritumor region was assessed based on the proportion of positive T cells among nucleated cells in the peritumoral area, which was described in previous studies (21 (link), 22 (link)).

Zhang S., Xu Y., Zhao P., Bao H., Wang X., Liu R., Xu R., Xiang J., Jiang H., Yan J., Wu X., Shao Y., Liang J., Wu Q., Zhang Z., Lu S, & Ma S. (2021). Integrated Analysis of Genomic and Immunological Features in Lung Adenocarcinoma With Micropapillary Component. Frontiers in Oncology, 11, 652193.

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Immunohistochemical Analysis of Tumor Markers

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Sections from the TMA blocks were cut at a thickness of 4 µm and stained using an automated immunostainer (BenchMark, Roche, Tucson, AZ, USA), with antibodies against PD-L1 clone 22C3 (Dako, Carpenteria, CA, USA), MLH1 (mouse monoclonal, clone M1, pre-diluted), MSH2 (mouse monoclonal, clone G219–1129, pre-diluted) and p53 (mouse monoclonal, Genemed, Torrance, CA, USA) according to the manufacturer’s instructions.

Arafa M., Shebl A.M., Salama A., ElZahaf E., Ashamallah S.A., Foda A.A., Awad A.E, & Shalaby A. (2022). Correlation of PD-L1 immunohistochemical expression with microsatellite instability and p53 status in endometrial carcinoma. European Journal of Obstetrics & Gynecology and Reproductive Biology: X, 16, 100172.

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