Methods for fluorescence histology have been published previously by our lab (5 (link)). Briefly, for mouse tumor xenografts, 5×106 NCI- N87 cells, purchased from ATCC, were injected in the rear flanks of 4–8 week-old female nude (Foxn1nu/nu) mice from Jackson Laboratories. Kadcyla was conjugated with AlexaFluor 680 NHS Ester (AF680, Thermo Fisher Scientific, A37567) with antibody to dye ratio of 0.3 or less and given intravenously. The mice were then sacrificed 24 hours after injection and histology slices were labeled ex-vivo with anti-mouse CD31 conjugated with AlexaFluor 555 (Thermo FisherScientific, A37571) and mouse antihuman IgG Fc antibody conjugated with AlexaFluor 488 (Thermo Fisher Scientific, A20000). All animal studies were conducted according to University of Michigan Institutional Animal Care and Use Committee.
Nci n87 cells
Manufactured by American Type Culture Collection
Sourced in United States
NCI-N87 cells are a human gastric carcinoma cell line derived from a metastatic gastric adenocarcinoma. They are adherent, epithelial-like cells and can be used for in vitro cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by System Biosciences (2 mentions)
Mouse anti cd31, by Thermo Fisher Scientific (1 mentions)
Foxn1 nu nu mice, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Ags cells, by American Type Culture Collection (1 mentions)
Antibiotic antimycotic abam, by Thermo Fisher Scientific (1 mentions)
Hi fbs, by Thermo Fisher Scientific (1 mentions)
Fbs hi, by Biowest (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Biowest (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Mkn 45 cells, by RIKEN Cell Bank (1 mentions)
Penicillin streptomycin, by Fujifilm (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Pc 9 cells, by European Collection of Authenticated Cell Cultures (1 mentions)
12 protocols using nci n87 cells
1
Fluorescence Histology of Xenograft Tumors
2
Cell Culture Conditions for Gastric Cancer
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AGS cells (ATCC CRL-1739) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% (v/v) FBS-HI (Gibco) and 1% (v/v) Antibiotic-Antimycotic (ABAM; Gibco) at 37°C in a humidified 5% CO2 incubator. NCI-N87 cells (ATCC CRL-5822) were cultured in RPMI 1640 (Gibco) supplemented with 10% (v/v) FBS-HI and 1% (v/v) ABAM. MKN28 cells stably transfected with an empty pRc/CMV expression plasmid as described previously [64 (link)] were a gift from Dr. Osamu Nagano (Division of Gene Regulation, Institute of Advanced Medical Research, Keio University School of Medicine). MKN28 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) FBS-HI (Biowest) and 1% (v/v) ABAM.
3
Investigating H. pylori-Mediated E-Cadherin Cleavage
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The gastric epithelial NCI-N87 cells (ATCC, CRL-5822) were grown in RPMI 1640 medium containing 4 mM glutamine (Invitrogen) and 10 % FCS (Sigma) in a humidified atmosphere at 37 °C. The H. pylori wild-type strain Hp26695 was cultured on agar plates containing 10 % horse serum under microaerophilic conditions for 48 h at 37 °C before infection experiments. Cells were infected with H. pylori at a MOI of 100 for 16 h. To investigate the influence of calcium ions or EGTA on H. pylori-mediated E-cadherin cleavage, infections were performed in the presence of indicated concentrations of calcium ions or EGTA. Cells were harvested in lysis buffer (20 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl, 1 % Triton X-100, 0.5 % DOC, 0.1 % SDS, 0.5 % NP-40). Samples were centrifuged for 10 min at 16,000×g at 4 °C to prepare whole cell lysates. Supernatants of infected cells were collected for the detection of the soluble extracellular E-cadherin fragment.
4
Gastric Cancer Cell Line Cultivation
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Two human gastric cancer cell lines were used in this study. HER2-expressing NCI-N87 cells were purchased from American Type Culture Collection (Manassas, VA), and HER2-negative MKN-45 cells were purchased from RIKEN Cell Bank (Tsukuba, Japan). Both cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10 fetal bovine serum and 1 % penicillin-streptomycin (Life technologies, Gaithersburg, MD) in a humidified incubator at 37 °C in an atmosphere of 95 air and 5 % carbon dioxide (CO2). The study was carried out in accordance with the Helsinki declaration.
5
Cell Culture of NCI-N87 and PC-9
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NCI-N87 cells (American Type Culture Collection [ATCC], Manassas, VA, USA) and PC-9 cells (The European Collection of Authenticated Cell Cultures [ECACC], Salisbury, UK) were cultivated in RPMI-1640 (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (System Biosciences, Palo Alto, CA, USA) and 1% penicillin-streptomycin (FUJIFILM Wako, Osaka, Japan), at 37 °C under 5% CO2.
6
Cell Culture and Protein Production
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NCI-N87 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and OE-19 cells were obtained from the European Collection of Cell Culture (ECACC, Porton Down, UK). The cell culture medium was RPMI-1640 supplemented with 10% fetal bovine serum (FBS), and antibiotics and cells were cultured at 37°C under 5% CO2. Trastuzumab and palivizumab was produced by Genentech (South San Francisco, CA, USA) and MedImmune, LLC (Gaithersburg, MD, USA), respectively. ChromPure human IgG (Jackson ImmunoResearch, West Grove, PA, USA) was used as human IgG control antibody for in vitro assays. IgG antibodies were produced using the Freestyle 293 system (Invitrogen, Carlsbad, CA, USA) and purified using protein-A affinity chromatography (GE Healthcare, Piscataway, NJ, USA). Endotoxin was removed with an Endotoxin Removal Kit (GenScript, Piscataway, NJ, USA), and endotoxin levels were determined using an Endotoxin Detection Kit (GenScript). Recombinant proteins were produced as secreted proteins using the Freestyle 293 system and purified using protein-A or Ni-NTA chromatography (Qiagen, Valencia, CA, USA) for Fc-tagged or His-tagged proteins, respectively.
7
NCI-N87 Cell Culture Protocol
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NCI‐N87 cells were purchased from the American Type Culture Collection, and were authenticated by short tandem repeat profiling and tested negative for mycoplasma. The cell line was cultured in RPMI‐1640 medium supplemented with 10% heat‐inactivated fetal bovine serum.
8
Gastric Cancer Cell Migration Assay
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MGC-803 and NCI-N87 cells were purchased from American Type Culture Collection. Dulbeco's Modified Eagle's Medium (DMEM) was purchased from Hyclone (Logan, UT, USA), fetal bovine serum, Lipofectamine 2000, and transwell chambers were purchased from Invitrogen (Carlsbad, CA, USA), Matrigel was purchased from BD Biosciences (NY, USA), CDX2, PTEN, E-cadherin, N-cadherin, GAPDH, and immunoglobulin G (IgG) antibodies were purchased from Santa Cruz Biotechnology (CA, USA), phosphorylation of Akt (pAkt) was purchased from Abcam (Cambridge, MA USA), immobilon western chemilumihescent horseradish peroxidase (HRP) substrate was purchased from Millipore (Billerica, MA, USA), and DAPI was purchased from Sigma (St. Louis, MO, USA).
9
Cell Line Culture Conditions
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SKBR3 cells were obtained from American Type Culture Collection and cultured at 37 °C, 5% CO2 in McCoy’s 5A (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA). BT-474 cells were obtained from American Type Culture Collection and cultured at 37 °C, 5% CO2 in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, Gibco) containing 10% FBS (Hyclone). NK92-CD16 cells were obtained from Biogen (Cambridge, MA, USA) and cultured at 37 °C, 5% CO2 in Myelocult H5100 (STEMCELL Technology, Vancouver, Canada) containing human interleukin (IL)-2 (Cell Signaling Technology) and puromycin (Gibco). NCI-N87 cells were obtained from American Type Culture Collection and cultured at 37 °C, 5% CO2 in RPMI 1640 (Gibco) containing 5% FBS (Hyclone). The MDA-MB-175-VII cell line for HER2/HER3 dimerization was obtained from American Type Culture Collection and cultured at 37 °C, 5% CO2 in RPMI 1640 (Gibco) containing 1% FBS (Hyclone).
10
Knockdown and Overexpression of ASPM and FOXM1
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AGS and NCI-N87 cells (American Type Culture Collection) were maintained as suggested by the provider. The 1 3
sustained knockdown (KD) of ASPM expression including all its putative transcript variants was achieved by lentivirusmediated RNA interference (RNAi) using validated small hairpin RNA (shRNA) oligonucleotides in the lentivector pLKO.1-puro (TRCN0000118905 and SHC002V [nontarget control]; Sigma-Aldrich, St. Louis, MO, USA). The specific KD of ASPM variant 1 (ASPMv1) expression, which encodes ASPMiI (NCBI RefSeq: NP_060606.3) was carried out by lentivirus-mediated transduction of small hairpin RNA (shRNA) using 2 independent RNAi target sequences (shRNA #4 and shRNA #3) specific for the exon 18 of the ASPM gene (unique to ASPMv1) and have been described previously [20] . The sustained KD of FOXM1 expression was achieved by lentivirus-mediated RNA interference (TRCN0000015545 and TRCN0000015544, MISSION shRNA lentiviruses; Sigma-Aldrich). The lentivirus-mediated overexpression (OE) vectors of dishevelled 3 (DVL3) and FOXM1 were purchased from Origene Technologies Inc. (Rockville, MD, USA). Lentivirus was produced as previously described [20] .
sustained knockdown (KD) of ASPM expression including all its putative transcript variants was achieved by lentivirusmediated RNA interference (RNAi) using validated small hairpin RNA (shRNA) oligonucleotides in the lentivector pLKO.1-puro (TRCN0000118905 and SHC002V [nontarget control]; Sigma-Aldrich, St. Louis, MO, USA). The specific KD of ASPM variant 1 (ASPMv1) expression, which encodes ASPMiI (NCBI RefSeq: NP_060606.3) was carried out by lentivirus-mediated transduction of small hairpin RNA (shRNA) using 2 independent RNAi target sequences (shRNA #4 and shRNA #3) specific for the exon 18 of the ASPM gene (unique to ASPMv1) and have been described previously [20] . The sustained KD of FOXM1 expression was achieved by lentivirus-mediated RNA interference (TRCN0000015545 and TRCN0000015544, MISSION shRNA lentiviruses; Sigma-Aldrich). The lentivirus-mediated overexpression (OE) vectors of dishevelled 3 (DVL3) and FOXM1 were purchased from Origene Technologies Inc. (Rockville, MD, USA). Lentivirus was produced as previously described [20] .
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