Cell lines were plated as 500 cells/well in 48-well plates (MatTek, 6mm, glass-bottomed), embedded in 9μL of phenol-red free matrigel matrix (Corning). Organoids were allowed to form for three weeks with media changes every 3-4 days. Live/dead imaging was performed by staining with Hoechst (5μg/mL), Calcein AM (1μM), and SYTOX Orange (0.5μM) for 1 hour prior to imaging with an Olympus IX81 inverted microscope. Relative ATP levels were measured using CellTiter Glo 3D (Promega). Fully-mature spheroids were formalin-fixed, paraffin embedded and sectioned. Sections were stained for hematoxylin and eosin (H&E) and imaged on a Zeiss Axio Scan.Z1. Quantification of hollow spheroids was done by a blind count of hollow spheroids by visual inspection from four equally sized regions of each H&E stained slide, analyzing only spheroids that were at least 50μm across.
Matrigel matrix phenol red free
Manufactured by Corning
Sourced in United States
Matrigel Matrix Phenol Red-Free is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is free of phenol red, a pH indicator commonly found in cell culture media. The matrix is a complex mixture of extracellular matrix proteins, including laminin, collagen IV, heparan sulfate proteoglycans, and entactin.
Automatically generated - may contain errors
Lab products found in correlation
Cycloheximide (chx), by Merck Group (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Mg132, by Peptide Institute (2 mentions)
E z bci hydrochloride, by Merck Group (2 mentions)
Ubiquitin, by R&D Systems (2 mentions)
Protease inhibitor mix, by Roche (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Mln4924, by Active Motif (2 mentions)
Recombinant human egf protein, by R&D Systems (2 mentions)
Spectramax spectrophotometer, by Molecular Devices (2 mentions)
Phosstop phosphatase inhibitor cocktail tablets, by Merck Group (2 mentions)
Axio scan z1, by Zeiss (1 mentions)
Ix81 inverted microscope, by Olympus (1 mentions)
Celltiter glo 3d, by Promega (1 mentions)
Primovert, by Zeiss (1 mentions)
Axiocam erc5s camera, by Zeiss (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
7 protocols using matrigel matrix phenol red free
1
3D Culture of Tumor Spheroids
2
FOXM1 Regulation of Endothelial Angiogenesis
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HTR8/SVneo cells transiently transfected with either FOXM1 siRNA or control NS siRNA, or pcDNA3-FOXM1 or empty vector EV-pcDNA3, under different oxygen tensions were resuspended in the RPMI medium with 2% FBS or in the endothelial growth media-2 (EGM-2, Bullet Kit, Lonza, Verviers, Belgium) (positive control) and seeded in triplicate in 96-well plates pre-coated with 70 μL growth factor reduced phenol-red free MATRIGEL matrix (Corning Life Sciences, Union City, CA, USA). Cells were incubated at 37 °C and under 3 or 1% O2 (accordingly) for 6 h and tube-like structures were examined with a phase-contrast microscope Primo Vert (Zeiss, Jena, Germany). One image per well was captured using an AxioCam ERc5s camera (Zeiss, Jena, Germany). Number of nodes, junctions, and meshes were analyzed using ImageJ-Angiogenesis Analyzer-HUVEC Phase Contrast software v1.0.c.
3
Matrigel Angiogenesis Assay with UDCA
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BALB/c nu/nu mice were administered a subcutaneous injection of 400 μL mixture containing 2 × 106 EA.hy 926 cells premixed with a Corning Matrigel Matrix phenol red-free (Cat. No. 356237), IL-8 (100 ng/ml), and UDCA (50 μM). Nude mice were randomly divided into three groups (the Control group, IL-8 group, and IL-8+UDCA group). Matrigel plugs were removed and imaged after 10 days. A Bestbio reagent kit (Bestbio, CHA) was applied to detect the hemoglobin content of the matrigel plugs according to the manufacturer’s protocol. Plugs were also snap frozen in the presence of optimum cutting temperature medium before sectioning and stained by hematoxylin and eosin (HE). Immunohistochemistry (IHC) assay was also performed to determine the expression of CD31, VEGF, and vWF (Santa Cruz Biotechnology, CA). Guide for the Care and Use of Laboratory Animals of the National Institutes of Health was strictly complying with in the in vivo experiment which was approved by the Committee on the Ethics of Animal Experiments of the Second Military Medical University.
4
3D Prostate Cancer Sphere Formation
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For sphere formation assay, an ultralow/non-attachment Petri dish (60 mm × 15 mm) was seeded with 4000 cells of stable EV and JICD-overexpressing PC cells (C4-2 and CWR22Rv1). Both attached and non-attached tumor spheres (TSs) were maintained for 7 days. Fresh media were added every 2 days. TSs were collected and analyzed for the expression levels of JICD, AR-Vs, and PC stemness-related markers.
A modified 3D cell culture was prepared with Matrigel® Matrix Phenol Red-free (#356231, Corning, Glendale, AZ, USA). Stable EV and JICD-overexpressing PC C4-2 and CWR22Rv1 cell suspensions containing 100 cells were mixed at 1:1 with cold Matrigel. Cell droplets were generated by pipetting 20 μL of droplets onto a 60 mm × 15 mm ultralow attachment Petri dish. The cell droplets were then incubated in a 5% CO2 incubator at 37 °C for at least 1 h to allow the Matrigel to solidify. Complete media (5 mL) were added to cover all droplets, which were maintained for 10 days. Fresh media were added every 2 days.
Spheres were imaged by using EVOS® FL Cell Imaging System (Thermo Fisher Scientific, Waltham, MA 02451, USA) at 10× and 20× magnification.
A modified 3D cell culture was prepared with Matrigel® Matrix Phenol Red-free (#356231, Corning, Glendale, AZ, USA). Stable EV and JICD-overexpressing PC C4-2 and CWR22Rv1 cell suspensions containing 100 cells were mixed at 1:1 with cold Matrigel. Cell droplets were generated by pipetting 20 μL of droplets onto a 60 mm × 15 mm ultralow attachment Petri dish. The cell droplets were then incubated in a 5% CO2 incubator at 37 °C for at least 1 h to allow the Matrigel to solidify. Complete media (5 mL) were added to cover all droplets, which were maintained for 10 days. Fresh media were added every 2 days.
Spheres were imaged by using EVOS® FL Cell Imaging System (Thermo Fisher Scientific, Waltham, MA 02451, USA) at 10× and 20× magnification.
5
Establishment and Propagation of Neuroblastoma PDXs
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Patient-derived xenografts (PDXs) were established from freshly obtained human stage 4 neuroblastoma tumor samples provided by the Biobank of the Virgen del Rocío Hospital in Seville. Relevant clinical information and characteristics are presented in Supplementary Table S1 . A 3 × 3 mm-sized fragment of the tumor was immediately obtained and placed with 30 μL of cold undiluted Matrigel (Corning Matrigel Matrix Phenol Red-Free, LDEV-Free. Ref: 356237, Corning Inc. New York, NY, USA) on a subcutaneous pocket performed on C.B-17 SCID mice (Harlan Laboratories, Indianapolis, IN, USA). Engrafted tumors were maintained in mice and monitored until they reached a volume of 1 cm3, then the tumor corresponding to the first passage of PDX (PDX1) was collected. A sample of this PDX1 was fixed in 4% PFA and embedded in paraffin for subsequent immunohistochemical study. The remaining tumor tissue was minced into 3 × 3 mm-sized fragments and freshly transferred to new recipient mice to generate the second passage of PDX (PDX2). This process was repeated until 5 serial in vivo passages were achieved. Engrafted mice were routinely checked for tumors and other signs of malignancy at sacrifice.
6
Protein Interaction Profiling by Mass Spectrometry
7
Protein Interaction Profiling by Mass Spectrometry
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Immunoprecipitation, immunoblotting experiments and Mass Spectrometry analysis were performed as previously described (Lignitto et al., 2019 (link)). Briefly, whole-cell lysates were generated by lysing cells in 50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP40, 1 mM EDTA, 10% glycerol, protease inhibitor mix (Roche) and phosphatase inhibitor cocktail (Sigma). Total protein amount was measured using a Spectramax spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Doxycycline (Sigma-Aldrich) was used at 0.1 μg/mL, Cycloheximide (Sigma-Aldrich) was used at 50 μg/mL, MLN4924 (Active Biochem) was used at 2 μM and MG132 (Peptides International) was used at 10 μM. (E/Z)-BCI hydrochloride, PhosSTOP phosphatase inhibitor cocktail tablets were purchased from Sigma-Aldrich. Matrigel Matrix Phenol Red-Free was purchased from Corning. Recombinant Human EGF Protein was obtained from R&D Systems. Purified E1, E2s and ubiquitin were purchased from Boston Biochem (now R&D Systems).
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