All antibody-dependent cellular assays were performed in the presence or absence of 5 µg/mL cetuximab (anti-EGFR, Merck KGaA) or trastuzumab (anti-HER2/neu, Roche) and with or without addition of 5 µg/mL SIRPα blocking antibody (clone 12C44 (link)). For the experiments with or without extracellular Ca2+, neutrophils and tumor cells were taken up in HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) supplemented with 5 g/L human albumin (Albuman, Sanquin Plasma Products) and 5.5 mM glucose or Minimal Essential Medium (MEM, Gibco), both with or without 1 mM CaCl2. For the experiments with the cell permeant chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N, N, N’, N’-tetraacetic acid), tumor cells were pretreated with BAPTA (ThermoFisher) for 30 min at 37°C and washed before coincubation with neutrophils. For the MEMbrane-cholesterol depletion experiments, tumor cells were preincubated with 2.5 mM methyl-β-cyclodextrin (MβCD) (Sigma) for 20 min at 37°C and washed before coincubation with neutrophils.
Anti her2 neu
Manufactured by Roche
Sourced in Germany
Anti-HER2/neu is a lab equipment product that detects the presence of HER2/neu protein in biological samples. It provides quantitative measurement of HER2/neu expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Bapta, by Thermo Fisher Scientific (1 mentions)
Methyl β cyclodextrin mβcd, by Merck Group (1 mentions)
Anti egfr, by Merck Group (1 mentions)
Histogel, by Thermo Fisher Scientific (1 mentions)
Clone 1e2, by Roche (1 mentions)
Clone 4b5, by Roche (1 mentions)
Clone sp1, by Roche (1 mentions)
Benchmark xt system, by Roche (1 mentions)
Anti pd l1, by Roche (1 mentions)
Zytolight spec erbb3 cen12 dual color probe, by ZytoVision (1 mentions)
5 protocols using anti her2 neu
1
Antibody-Dependent Cellular Cytotoxicity Assay
2
Evaluating HER2 Status in Gastric Cell Lines
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The HER2 status in the cell lines MKN1, MKN7, and NCI‐N87 was evaluated by immunohistochemistry (IHC). Cells were seeded with a density of 2 × 104 cells·cm−2 for MKN1 and MKN7, and 2.4 × 104 cells·cm−2 for NCI‐N87. After 24 h, the cells were washed, scraped off, and fixed with 4% formalin. The next day, the cells were centrifuged and the pellet was resuspended in a smaller volume of formalin. Richard‐Allan Scientific™ HistoGel™ was then added to the cell pellet. After an appropriate cooling time, the mixture block was transferred into a histocassette and embedded in paraffin. Slices were prepared, and the antibody anti‐HER‐2/neu (Roche Penzberg, Germany; #4B5) was used for IHC.
3
Breast Cancer Biopsy and Biomarker Analysis
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Tumor core biopsies were performed before NAC therapy commenced. ER, PgR, Ki-67 and HER2 were assessed by IHC. IHC staining for ER (Confirm anti-ER, rabbit monoclonal primary antibody; clone SP1, Ventana Medical Systems), PR (Confirm anti-PR, rabbit monoclonal primary antibody; clone 1E2; Ventana Medical Systems), ki-67 (mouse monoclonal primary antibody; clone MIB1; Origene), and HER2 (anti-HER2/neu, rabbit monoclonal primary antibody; clone 4B5; Ventana) was performed.
The percentage and intensity of nuclear staining with ER and PR were estimated, and nuclear staining of ≥ 1% of the invasive tumor nuclei was interpreted as positive, in accordance with 2010 ASCO/CAP guidelines. HER2 positivity refers to cases with an IHC score of 3 + or fluorescence in situ hybridization (FISH) amplification (by 2013 ASCO/CAP criteria) for IHC scores of 2+. Patients were separated into three groups for the purposes of our analysis according to ER expression level: ER-negative, ER low positive (ER staining 1-10%), and ER > 10% positive (ER staining > 10%). The ER expression status on the surgical specimen was recorded. The assessments were performed locally by two experienced pathologists.
The percentage and intensity of nuclear staining with ER and PR were estimated, and nuclear staining of ≥ 1% of the invasive tumor nuclei was interpreted as positive, in accordance with 2010 ASCO/CAP guidelines. HER2 positivity refers to cases with an IHC score of 3 + or fluorescence in situ hybridization (FISH) amplification (by 2013 ASCO/CAP criteria) for IHC scores of 2+. Patients were separated into three groups for the purposes of our analysis according to ER expression level: ER-negative, ER low positive (ER staining 1-10%), and ER > 10% positive (ER staining > 10%). The ER expression status on the surgical specimen was recorded. The assessments were performed locally by two experienced pathologists.
4
Immunostaining Evaluation Protocol for Tumor Markers
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Immunostaining was performed using a Roche Benchmark XT system and anti-CD3 (Clone 2GV6, Ventana - Roche), anti-CD8 (Clone SP57, Ventana - Roche), anti-HER2/Neu (Clone 4B5, Ventana - Roche) and anti-PD-L1 (Clone SP263, Ventana - Roche) antibodies. Immunostaining were evaluated by two independent qualified pathologists. In four cases of discrepancy, an additional assessment was performed by a third senior pathologist. For CD3 and CD8, average values were obtained from examining all intra and peritumoral areas. A semi-quantitative score was defined; CD3 and CD8 expression was classified according to the percentage of total tumor-related lymphocyte (peritumoral and intratumorally) staining: low (0–34%), moderate (35–64%) and high (65–100%). PD-L1 was evaluated using the Combined Positive Score (CPS) established for gastric/gastroesophageal junction adenocarcinoma [22] . HER2/Neu scoring was performed according to the College of American Pathologists (CAP), which describes three categories: HER2-negative (0;1+), HER2-equivocal (2+) and HER2-positive (3+) [23] (link).
5
Immunohistochemistry and FISH for HER2, HER3, and PD-L1
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Immunohistochemical examinations were performed using monoclonal rabbit antibodies against the HER-2 protein (clone 4B5, prediluted, Ventana anti-HER2/neu), HER-3 (clone HER-3/c-erbB-3RMab) and PD-L1 (Anti-PD-L1, Clone28-8, Dako). Automated HER-2, HER-3 and PD-L1 staining was conducted by the immu nostainer BenchMark Ultra following the manufacturer's instructions, with an ultraView Universal DAB Detection kit and bluing reagent as a visualization reagent and chro mogen. All materials were obtained from Roche Diagnostics GmbH, Mannheim, Germany.
Fluorescence in situ hybridization (FISH). Sections (thickness of 2-3 μm) of paraffi n-embedded tissues were processed for FISH using materials as follows: ZytoLight SPEC HER2/CEN 17 Dual Color probe and ERBB3 gene amplifi cation ZytoLight SPEC ERBB3/CEN12 Dual Color Probe (ZytoVision GmbH, Bremerhaven, Germany). The assay procedures involving materials precisely followed the manufacturer's instructions.
Fluorescence in situ hybridization (FISH). Sections (thickness of 2-3 μm) of paraffi n-embedded tissues were processed for FISH using materials as follows: ZytoLight SPEC HER2/CEN 17 Dual Color probe and ERBB3 gene amplifi cation ZytoLight SPEC ERBB3/CEN12 Dual Color Probe (ZytoVision GmbH, Bremerhaven, Germany). The assay procedures involving materials precisely followed the manufacturer's instructions.
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