A total of 2 × 104 cells were seeded per well of 96-well plate (Nunclon delta surface, Thermo Scientific, #167008) to reach confluent monolayer. Scratches were made with Woundmaker 96 (Essen, Bioscience) and imaging was performed with IncuCyte Live Cell Imaging System (Essen, Bioscience). The recorded images of the scratches were analyzed with IncuCyte software to quantify gap closure. For invasion assay transwell chambers with 8 μm pore size (Thermo Scientific Nunc) were coated with matrigel matrix from Corning (Thermo Scientific). Then cell suspension of 1 × 105/300 µL in RPMI 1640 supplemented with 0.1% FBS was added to the matrigel coated upper chamber and the medium containing 10% FBS was added to the lower chamber as a chemoattractant. Cell were allowed to invade for 48 hr after which the cells which migrated to the other side of the membrane were fixed with 4% PFA and stained with DAPI. Images were acquired using QImaging (Pecon, software Micro-Manager 1.4.22) with 10x magnification, and the cells were counted using Image J software.
Matrigel matrix
Manufactured by Thermo Fisher Scientific
Sourced in United States
Matrigel matrix is a gelatinous protein mixture that is extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is commonly used as a basement membrane extract to support the growth and differentiation of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel matrix, by BD (4 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
E0771, by American Type Culture Collection (2 mentions)
Metamorph image analysis software, by Molecular Devices (2 mentions)
24 well insert, by Corning (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Matrigel matrix, by Corning (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Wound maker 96, by Sartorius (1 mentions)
Nunclon delta surface, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Collagenase 1, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Countess 2 cell counter, by Thermo Fisher Scientific (1 mentions)
Sb431542, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
35 protocols using matrigel matrix
1
Quantifying Cell Migration and Invasion
2
Tumor Latency in SV40 Transgenic and Syngeneic Breast Cancer Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used tumor latency as the primary outcome for SV40 transgenic mouse model and tumor incidence was measured weekly. The end point for SV40 mice was 20 weeks based on our previous studies [14 (link),20 ] and the instruction provided by the Jackson Laboratory. Because wild-type C57 mice are phenotypically normal, 5 randomly selected C57 female offspring mice were injected with the mixture of ~106 murine mammary tumor cells, E0771 (ATCC, Manassas, VA, USA), and same volume of Matrigel Matrix (Thermo Fisher Scientific, Wilmington, DE, USA) under mammary pads at 21 weeks to bear syngeneic breast tumor as done previously [14 (link),15 (link)]. All C57 offspring mice including 5 engrafted and the rest tumor-free mice were sacrificed 4 weeks post injection at 25 weeks of age. We used tumor weight as the primary outcome and tumor volume was measured weekly in C57 mice. Tumor volumes were measured by a caliper weekly and calculated using the formula: tumor volume (cm3) = (length × width2) × 0.523 as described previously [14 (link),15 (link),26 (link)]. At the endpoint, the mammary tumors were collected, weighed and used for further analyses.
3
Angiogenic Potential of HRMECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The angiogenic activity of HRMECs was determined using a Matrigel tube formation assay. Briefly, after transfection and exposure conditions, HRMECs were plated at a density of 30 000 cells/well in 96-well plates precoated with 50 μl of growth factor reduced Matrigel Matrix (Thermo Fisher Scientific, New Hampshire, United States) and cultured at 37°C for 6 h in normoxia or hyperoxia in complete endothelial growth medium (see endothelial cell culture section), or PAC-CM pre-subjected or not hyperoxia, or si-PTPN9-treated-PAC-CM. Capillary-like tubes were observed under a light microscope. Images were obtained at 10× magnification, and all tubes and branching point were counted.
4
Murine Tumor Organoid Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumors were excised from mice and dissociated using 2 mg/ml collagenase I (Thermo Fisher Scientific). Resulting dissociated tissue and cells were filtered using a 70 μm nylon cell strainer and centrifuged at 250g for 5 min at room temperature. Viability and concentration were assessed using a Countess II Cell Counter (Thermo Fisher Scientific). Cells were plated at 50,000 live cells in 15 μl Matrigel Matrix (Thermo Fisher Scientific). Once Matrigel was solidified, cells were cultured with organoid media consisting of 50% advanced Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific), 50% L-WRN conditioned media (American Type Culture Collection [ATCC] CRL-3276; both supplemented with 20% fetal bovine serum (FBS) (Sigma), 2 mM GLUTamax, 100 units/ml penicillin and 0.1 mg/ml streptomycin, 10 μM SB 431542, 10 μM Y-27632, and 50 μg/ml gentamicin (all Thermo Fisher Scientific)). Media were changed every other day.
5
Cell Migration and Invasion Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transwell migration and invasion assays were carried out using a chamber containing a polycarbonate filter with a pore size of 8.0 μm (24-well insert; Corning, NY, USA). Polycarbonate filters precoated with Matrigel Matrix (BD Biosciences, San Jose, CA, USA) were used for the invasion assay, and uncoated filters were used for the migration assay. Cells were serum-starved for 8 h. The lower chambers were filled with 800 mL of MEM (SiHa/C-33A) or DMEM (HeLa) supplemented with (concentrations of 2.5–20 mM) or without PLA. Approximately 3–5 × 104 cells were resuspended in 300 μL of serum-free medium and added to the upper chamber. After incubation for 16 h at 37 °C and 5% CO2, the lower chambers were incubated with calcein-AM (Thermo Fisher Scientific) to stain the cells that invaded through the uncoated membrane or the Matrigel Matrix. The stained cells were counted in 4 randomly selected visual fields using a CCD camera mounted to an inverted microscope running MetaMorph image analysis software (Molecular Devices, Sunnyvale, CA).
6
Culturing Human Intestinal Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. As described previously, human intestinal organoid cultures and passages were conducted (40 ). Briefly, organoids were cultured with a 25% L-WRNH–conditioned medium embedded in Matrigel Matrix (Corning). The entire medium was changed every 2 to 3 days, and passages were performed every 5 to 6 days. Following passages, cell dissociation was performed using TrypLE express (Corning) at a general ratio of 1:8 embedded in Matrigel Matrix on a Nunclon Delta 4-well dish (Thermo Scientific). All cells were cultured at 37 °C in a 5% CO2 atmosphere. The experiments using primary human organoids complied with the Declaration of Helsinki and were approved by the human ethical committee of The University of Tokyo and Osaka University. All tissues were sampled with informed consent.
7
Subcutaneous and Orthotopic Tumor Transplantation in Pak4 Knockout Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the subcutaneous tumor transplantation model, 1 × 106 Hepa 1–6 cells were resuspended in 100 μl Matrigel matrix (1:1 with PBS, # CLS354234, Thermo Fisher Scientific) and injected subcutaneously onto both flanks of 8-week-old male Pak4 LKO or WT mice. Once a tumor had grown to 500 mm3, the tumor-bearing mouse was fed either an NCD or KD for 9 days. Tumor size was measured with electronic calipers and volumes were determined using the following formula: length (mm) × width (mm)2 × 0.5. Mice were sacrificed when their tumors had grown to ~2000 mm3. For the orthotopic tumor transplantation model, 1 × 106 Hepa 1–6 cells were directly injected into the livers of WT and Pak4 LKO mice. One week later, mice were fed with either an NCD or KD for an additional 2 weeks.
8
Tumor Latency in SV40 Transgenic and Syngeneic Breast Cancer Mice
9
Human Pluripotent Stem Cell Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human ESCs employed in this study were derived from blastocyst-stage embryos obtained with the informed consent from the donors. A well-characterized hESC line [32 (link)], CCTL14 (Center of Cell Therapy Line 14), was used in passages 29-41. For human iPSCs, the well-characterized line ID CBIA-19, derived in our laboratory from human umbilical vein endothelial cells using genome nonintegrating vectors [33 (link)], was used from passages 34 to 41. All hPSCs were grown on a Matrigel™ matrix (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in mTESR™1 (Stemcell Technologies, Vancouver, Canada). To induce differentiation, hPSCs were cultured in DMEM/F12 (Life Technologies, Carlsbad, California, USA) containing the GSK3β inhibitor CHIR 99021 (4 μM) (Sigma-Aldrich, St. Louis, Missouri, USA) for three consecutive days.
10
Matrigel Invasion Assay for Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transwell inserts (Millipore, Billerica, MA, USA) containing polycarbonate filters with 8-µm pores were used in the assay. The inserts were coated with 50 µl of Matrigel matrix (1 mg/ml) according to the manufacturer's recommendations (Thermo Fisher Scientific, Inc.). The cells were seeded in the upper chambers of the inserts at a density of 2×105 cells in 1 ml serum-free MCF-10A medium. MCF-10A medium (2 ml) containing serum was placed in the lower chambers. Following 72 h of treatment, the cells on the upper surface of the membrane were removed using a methanol coated cotton swab. The cells on the lower chamber were fixed in 4% paraformaldehyde and stained with hematoxylin (Sigma). For each membrane, the number of cells was counted in 10 evenly distributed ×40 fields of view using a light microscope (Nikon Eclipse 80i). Each experiment was repeated in triplicate.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!