The KPC-derived PDAC cell line (TB32048) previously generated from a female C57BL/6 KPC mouse was provided as a generous gift from David Tuveson laboratory. TB32048 was cultured for 3–4 passages at maximum 80% confluency in 10% fetal calf-serum (#A15-104, GE Healthcare) in DMEM (#E15-810, PAA) + 100 μg/ml penicillin/streptomycin (#P11-G10, PAA) in a T175 flask in standard conditions (37°C, 5% CO2) and tested regularly in house for mycoplasma (#rep-pt1, Invivogen and #LT07-710, Lonza). Cells were dissociated using 0.1% trypsin (PBS) (#594-18C, Sigma) for 10 min at 37°C and resuspended in PBS and BD Matrigel™ Basement Membrane Matrix High Concentration (#354248, BD Biosciences) in a ratio of 1:1. 1,000 cells in 5 μl was injected into the pancreas using a Hamilton® syringe, 700 series (#10100332, Fisher Scientific). For the experiments used to assess CD138hi cells and anti-CD20 at day 10, 10,000 cells in 30 μl Matrigel, or Matrigel only sham surgery was performed. The peritoneal wall was sutured using 6/0-gauge coated vicryl sutures™ (#W9500T, Ethicon) and skin closed using two 9 mm Clay Adams Clips (#IN015A, VetTech Solutions) and an Autoclip® applier (#IN015B, VetTech Solutions).
Matrigel basement membrane matrix high concentration
Manufactured by BD
Sourced in Italy
BD Matrigel™ Basement Membrane Matrix High Concentration is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is a complex mixture of extracellular matrix proteins including laminin, collagen IV, heparan sulfate proteoglycans, and entactin.
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Lab products found in correlation
3 protocols using matrigel basement membrane matrix high concentration
1
Orthotopic Implantation of KPC-derived PDAC Cells
2
Docetaxel Treatment of SCID Mice Xenografts
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Severe combined immunodeficient (SCID) mice of ages 5 weeks were purchased from Charles River Laboratories and maintained in a temperature-controlled, pathogen-free room. Before the experiments, the animals were quarantined and acclimatized for two weeks. Mice were kept in community cages on a standard laboratory diet with free access to drinking water and a 12 h day/night regime. All animals were handled according to the approved protocols and guidelines of 2nd Local Ethics Committee for Experiments on Animals at the Jagiellonian University in Cracow (Dec No. 290/2017). Cancer cells were mixed with BD Matrigel basement membrane matrix high concentration (1:1 in PBS; BD Biosciences), and 40 μL of the cold liquid Matrigel-mixed (1.5 × 105) cells was slowly injected subcutaneously into abdominal flank of SCID mice. The mice were observed for 3 to 5 weeks for the appearance and development of tumors. Every 4 days, docetaxel (20 mg/kg) was administrated intraperitoneally to the mice. The tumor volume was calculated according to the following formula: V = (Π/6)a×b×c, where a, b, c are perpendicular diameters of the ellipsoid approximating the shape of the tumor.
3
Smad7 Antisense Oligonucleotide in Colorectal Cancer
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HCT-116 (5 × 105 cells) were resuspended in 500 μl BD Matrigel Basement Membrane Matrix High Concentration (BD Biosciences, Milan, Italy) and injected subcutaneously into the flank of 8-week-old female Rag1−/− mice, whose fur was shaved and depilated. After 1 week, mice with similar tumor volume, determined by caliper measurement, were divided into two groups and received daily intraperitoneal injection of either Smad7 sense oligonucleotide or Smad7 antisense oligonucleotide (both at 100 μg/mouse resuspended in 300 μl PBS). Mice were sacrificed at day 21. Tumors were excised, photographed, their volume calculated according to the following formula: 1/2 × (short diameter) × (long diameter) × (height) and finally included in OCT (Thermo Scientific, Rockford, IL, USA) for histochemistry experiments. To assess the effective uptake of the Smad7 antisense oligonucleotide in the growing tumors, in preliminary experiments mice received a single intraperitoneal injection of either FITC-conjugated Smad7 antisense oligonucleotide (used at 100 μg/mouse) or PBS (control). Twenty-four hours later mice were killed, tumors excised and FITC-conjugated Smad7 antisense oligonucleotide distribution assessed by immunofluorescence.
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