Trastuzumab (Herceptin®; Hoffman-La Roche, Mississauga, Ontario, Canada), T-DM1 (Kadcyla®; trastuzumab emtansine; Hoffman- La Roche) and IVIG (Gamunex®; 10% immune globulin intravenous (human), Grifols, Mississauga, Ontario, Canada, DIN 02247724) were obtained from clinical preparations at the Ottawa Hospital Pharmacy, stored at 4 °C and used at the indicated concentrations. Colchicine (Sigma-Aldrich, Cat. # C9754) was resuspended in 100% DMSO to 100 mM and was stored at −80 °C and diluted to 100 µM in DMSO before use. Recombinant human TNFα (R&D Systems, Oakville, Ontario, Canada, Cat. # 210-TA) was resuspended in sterile PBS with 0.1% BSA and stored at −20 °C. All compounds were diluted to specified conditions in serum-free media for all assays. For competition assays, 786-0 cells and JIMT1 cells were seeded in 24-well plates and incubated overnight at 37 °C in a 5% CO2 humidified incubator. Cells were then pretreated with trastuzumab or IVIG (786-0 at 1000 μg/ml, JIMT1 at 250 μg/ml), or mock-treated for 2 h. Next, supernatants were aspirated and cells were treated with equivalent concentrations of T-DM1 for 2 h. Subsequently, cells were washed once with PBS and infected with VSVΔ51-GFP at MOI 0.01 for 45 h.
Kadcyla
Manufactured by Roche
Sourced in Canada
Kadcyla is a type of laboratory equipment used for the analysis and processing of biological samples. It functions as a specialized device designed for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Herceptin, by Roche (3 mentions)
Gamunex, by Grifols (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Colchicine, by Merck Group (1 mentions)
Recombinant human tnf α, by R&D Systems (1 mentions)
Ammonium sulfate, by Merck Group (1 mentions)
Centrifugal filter tubes amicon 30 kda, by Merck Group (1 mentions)
Maleimidocaproyl valine citrulline monomethyl auristatin e vcmmae, by MedChemExpress (1 mentions)
Tween 20, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Isopropyl alcohol, by Merck Group (1 mentions)
Anti mouse cd31, by BioLegend (1 mentions)
Mouse antihuman igg fc antibody, by BioLegend (1 mentions)
Alexa fluor 680 nhs ester, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw nhs ester, by LI COR (1 mentions)
Alexa fluor 750, by Thermo Fisher Scientific (1 mentions)
Celltrace far red ddao se, by Thermo Fisher Scientific (1 mentions)
8 protocols using kadcyla
1
Trastuzumab, T-DM1, and IVIG Competition Assay
2
Analytical Workflow for Antibody-Drug Conjugates
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Ammonium sulfate, sodium phosphate, sodium chloride, tris(2-carboxyethyl) phosphine (TCEP), N-acetylcysteine (NAC), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME), ethylenediaminetetraacetic acid (EDTA), phosphate-buffered saline (PBS), and TWEEN® 20 were purchased from Sigma-Aldrich. Centrifugal filter tubes (Amicon-30 kDa) were purchased from Merck Millipore. N-Succinimidyl-4-(maleimidomethyl) cyclohexanecarboxylate-emtansine (SMCC-DM1) was obtained from ALB Technology. Kadcyla® was purchased from Roche. Maleimidocaproyl-valine-citrulline-monomethyl auristatin E (VCMMAE) was obtained from MedChem Express. Lithium dodecyl sulfate (LDS) sample loading buffer (4X) and 12% acrylamide PAGE gel were purchased from Invitrogen. NUNC 96-well Maxisorb immunoplates were purchased from Thermo Fisher Scientific.
3
Labeling Antibodies and ADCs for Imaging
4
Her2-Targeted Antibodies in Cancer
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The following antibodies were used: anti-Her2/ErbB2 rabbit mAb (Cell Signaling Technology, Danvers, MA, USA), anti-actin rabbit polyclonal antibody (Sigma-Aldrich), trastuzumab (Herceptin; Genentech, South San Francisco, CA, USA), pertuzumab (Perjeta®; Roche Diagnostic, Mannheim, Germany) and trastuzumab-DM1 (TDM1) (Kadcyla®; Roche).
5
Trastuzumab, T-DM1, and Paclitaxel Cytotoxicity
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Cells were seeded into 24 well plates at a density of 0.1 x 106 cells/ml and incubated alone or in 10 μg/ml trastuzumab (Herceptin, Roche Ltd., Basel, Swiss), 1 μg/ml T-DM1 (Kadcyla, Roche Ltd., Basel, Swiss), or 1μg/ml paclitaxel (Phyxol, Sinphar Ltd., I-Lan, Taiwan) for 72 hr [37 (link)]. At the end of this period, 0.1 ml of 0.4% trypan blue and deionized water (1:1) was added to the control and treated tubes to estimate the number of dead cells. Cell viability was estimated with a hemocytometer. Dead cells were stained blue, while live cells remained unstained. The cell mortality was expressed as the percentage of trypan blue-positive cells compared to the total number of cells. The viability percentage was presented by the number of live cells divided by the number of untreated control cells ×100.
6
EGF Stimulation of Serum-Starved Cells
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Trastuzumab (Herceptin®; Hoffman-La Roche, Mississauga, Ontario, Canada), T-DM1 (Kadcyla®; trastuzumab emtansine; Hoffman- La Roche) and IVIG (Gamunex®; 10% immune globulin intravenous (human), Grifols, Mississauga, Ontario, Canada, DIN 02247724) were obtained from clinical preparations at the Ottawa Hospital Pharmacy, stored at 4°C and used at the indicated concentrations. Human (R&D Systems, Cat. # 236-EG) or mouse (R&D Systems, Cat. # 2028-EG) recombinant EGF were used to stimulate serum-starved cells for 30 minutes prior to protein extraction.
7
Cytotoxicity Assay of Anticancer Agents
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Tumor cells were seeded as described above. After 24 h cells were treated with different concentrations of paclitaxel (#33069-62-4, Paclitaxel Hospira, Pfizer), doxorubicin (#23214-92-8, Farmiblastina, Pfizer), or T-DM1 (#1018448-65-1, Kadcyla, Roche). Treatment lasted for 3 days in the case of the chemotherapeutic agents paclitaxel and doxorubicin, and 6 days when treated with T-DM1. Cell death was assayed by crystal violet staining of alive cells and read at the Infinite M200 Pro Multimode Microplate Reader (TECAN).
8
Photodynamic Therapy for Breast Cancer
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Escherichia coli strain BL21(DE3) (catalog number: 312–06534, Nippon Gene) was used as an expression host. The pET‐45b(+) vector was used for cloning and gene expression analysis (catalog number: 71327; Novagen). The denaturation buffer consisted of 0.1 M Tris‐HCl, pH 8.5, 10 mM EDTA, and 6 M guanidine hydrochloride. The refolding buffer consisted of 0.1 M sodium phosphate and 0.4 M arginine‐HCl, pH 6.0. The gel filtration buffer consisted of 0.1 M sodium phosphate and 0.2 M arginine‐HCl, pH 6.5. The photosensitizer, Psyche‐Ax‐SiPc, was provided by Prof. Kanai's laboratory at the University of Tokyo.11 , 13 Human breast cancer KPL‐4 cells were a generous gift from Prof. Kurebayashi (Kawasaki Medical School). Kadcyla® (trastuzumab emtansine) was purchased from Roche.
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Escherichia coli strain BL21(DE3) (catalog number: 312–06534, Nippon Gene) was used as an expression host. The pET‐45b(+) vector was used for cloning and gene expression analysis (catalog number: 71327; Novagen). The denaturation buffer consisted of 0.1 M Tris‐HCl, pH 8.5, 10 mM EDTA, and 6 M guanidine hydrochloride. The refolding buffer consisted of 0.1 M sodium phosphate and 0.4 M arginine‐HCl, pH 6.0. The gel filtration buffer consisted of 0.1 M sodium phosphate and 0.2 M arginine‐HCl, pH 6.5. The photosensitizer, Psyche‐Ax‐SiPc, was provided by Prof. Kanai's laboratory at the University of Tokyo.
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