Gfr matrigel

Manufactured by Thermo Fisher Scientific

GFR Matrigel is a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is used as a substrate for culturing cells in vitro, and it can support the growth and differentiation of various cell types.

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Matrigel (702 citations)

5 protocols using gfr matrigel

VEGFA-Mediated Angiogenic Tube Formation

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After thawing overnight at 4°C, 250 μL Growth Factor Reduced (GFR) Matrigel (Thermo Fisher Scientific; #A1413202) was placed into each well of cold 24-well plates using cold pipette tips, and the plates were then placed at room temperature for 30 min. 4 × 10⁴ HCAECs were placed in each of the Matrigel-coated wells and incubated for 24 h under standard cell culture conditions (37°C, 5% CO₂). Wells containing PBS were used as controls. To analyze the dose-dependent rescue, three different exogenous VEGFA concentrations: 0 pg/mL, 500 pg/mL, and 5,000 pg/mL were used. Network formation was quantified by measuring the number of branches, number of loops, and total length of the tube. Digital images of microtiter well sections were obtained by using a Zeiss Axiovert 200M microscope and ZEN 2.3 Pro software. Data were analyzed with ImageJ image analysis software (NIH, USA).

Hosen M.R., Li Q., Liu Y., Zietzer A., Maus K., Goody P., Uchida S., Latz E., Werner N., Nickenig G, & Jansen F. (2021). CAD increases the long noncoding RNA PUNISHER in small extracellular vesicles and regulates endothelial cell function via vesicular shuttling. Molecular Therapy. Nucleic Acids, 25, 388-405.

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Establishment of Metastatic Renal Cell Carcinoma Organoids

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CT-guided biopsies were conducted to obtain mRCC lung metastases. The biopsies were conditioned in 5 mL PBS with 5 mM EDTA for 15 min at room temperature, then digested in 5 mL PBS with 1 mM EDTA and 2× TrypLe (Thermo Fisher Scientific) for 1 h at 37 °C. Following digestion, mechanical force (pipetting) was applied to facilitate cell release in solution. Dissociated cells were collected in Advanced DMEM/F-12 (Thermo Fisher Scientific), pelleted, resuspended in 120 µL growth factor reduced (GFR) Matrigel (Corning), and seeded in 24- or 48-well flat bottom cell culture plates (Corning). The Matrigel was then solidified for 20 min in a 37° C and 5% CO₂ cell culture incubator, and overlaid with 500 µL complete human organoid media. The complete media was refreshed every two days. Passaging of MDOs was performed using TrypLe. Briefly, MDOs were mechanically harvested (by pipetting) out of Matrigel using PBS with 1 mM EDTA and 1× TrypLe, then incubated for 20 min at 37 °C. MDOs were then dissociated to single cells by applying mechanical force (pipetting), washed with HBSS (Thermo Fisher Scientific), pelleted, resuspended in GFR Matrigel, and re-seeded at an appropriate ratio. Additionally, specific human MDO culture media were collected, which were made up of the specific constituents shown in Table S1.

Zhang C., Chen L., Liu Y., Huang J., Liu A., Xu Y., Shen Y., He H, & Xu D. (2021). Downregulated METTL14 accumulates BPTF that reinforces super-enhancers and distal lung metastasis via glycolytic reprogramming in renal cell carcinoma. Theranostics, 11(8), 3676-3693.

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Organoid-Stromal Cell Co-culture in Hypoxia

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Organoids were derived from pancreatic tumors of KPC (Kras^LSL-G12D/+;Trp53^LSL-R172H/+;Pdx1-Cre) mice in a C57BL/6 background and described before (23 ). Organoids were cultured 24-well plates in growth factor reduced (GFR) Matrigel (Corning) in Advanced DMEM/F12 supplemented with the following: 1% P/S, 2 mM glutamine, 1X B27 supplement (12634–028, Invitrogen), 50 ng/ml murine EGF (PMG8043, Peprotech), 100 ng/ml murine Noggin (250–38; Peprotech), 100 ng/ml human FGF10 (100–26; Peprotech), 10 nM human Leu-Gastrin I (G9145, Sigma), 1.25 mM N-acetylcysteine (A9165; Sigma), 10 mM nicotinamide (N0636; Sigma), and R-spondin1 conditioned media (10% final). Organoids were passaged with every 3–4 days. For PSC co-culture, confluent wells of organoids were dissociated with 1x TrypLE (12604013, Thermo Fisher) and plated at a splitting ratio of 1:5 (approximately 1×10⁴ cells) together with 8×10⁴αSMA-DsRed expressing PSCs in GFR Matrigel. Co-cultures were cultured with DMEM supplemented with 10% FBS (Gemini) and 1% P/S in 20% O₂ and 5% CO₂. For experiments in hypoxia, co-cultures were placed in a hypoxia glove box (Coy) set at 0.5% O₂ for the last 48h of the experiment. For measurement of organoid growth in co-cultures, organoids expressing Luciferase were used. Cultures were treated with Luciferin, lysed and luminescence was measured.

Zak E, & Pakrasi H.B. (2000). The BtpA Protein Stabilizes the Reaction Center Proteins of Photosystem I in the Cyanobacterium Synechocystis sp. PCC 6803 at Low Temperature. Plant Physiology, 123(1), 215-222.

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Isolation and Propagation of Pancreatic Organoids

Cited 2 times

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Detailed procedures to isolate and propagate mouse and human, normal and
neoplastic pancreatic organoids have been described previously [22 (link), 25 (link)]. In
brief, normal pancreatic mouse ducts were manually picked after enzymatic
digestion of pancreas with 0.012% (w/v) collagenase XI (Sigma) and 0.012% (w/v)
dispase (GIBCO) in DMEM media containing 1% FBS (GIBCO) and were seeded in
growth factor-reduced (GFR) Matrigel (BD). For tumors (mT), bulk tissues were
minced and digested overnight with collagenase XI and dispase and embedded in
GFR Matrigel. In the case of human primary and metastatic pancreatic tumor
organoid cultures (hT), tumor tissue was minced and digested with collagenase II
(5 mg/ml, GIBCO) in human complete medium at 37°C for a maximum of 16
hours. The material was further digested with TrypLE (GIBCO) for 15 minutes at
37°C, and embedded in GFR Matrigel. Normal samples were processed as
above, except that the collagenase digestion was done for a maximum of 2 hours
in the presence of soybean trypsin inhibitor (1 mg/ml, Sigma).

Ponz-Sarvise M., Corbo V., Tiriac H., Engle D.D., Frese K.K., Oni T.E., Hwang C.I., Öhlund D., Chio I.I., Baker L.A., Filippini D., Wright K., Bapiro T.E., Huang P., Smith P., Yu K.H., Jodrell D.I., Park Y, & Tuveson D.A. (2019). Identification of Resistance Pathways Specific to Malignancy Using Organoid Models of Pancreatic Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(22), 6742-6755.

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Maintenance of Human Induced Pluripotent Stem Cells

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The hiPSC line Edi042A was purchased from Cedars Sinai. The hiPSC line WTC-ZO1-mEGFP (AICS-0023)^{27 (link)} was a gift from the Gordana Vunjak-Novakovic lab. Both cell lines were maintained in six-well plates coated with Matrigel growth factor reduced basement membrane matrix (GFR-Matrigel, Corning, 354230) in mTeSR plus medium (STEMCELL Technologies, 100–0276). To coat the six-well plates, 1.5 mL of GFR-Matrigel (diluted at a 1:100 ratio with DMEM/F12 medium, Gibco, 11320033) was added per well and incubated at 37°C for 1h. To passage hiPSCs (60–70% confluency), cells were rinsed with 3–4 mL of DMEM/F12 medium per well, and then 2 mL of Accutase (Sigma-Aldrich, A6964) was added for 5–6 min at 37°C. Subsequently, 2 ml of mTeSR plus medium was added to neutralize the dissociation. Cells were collected after centrifugation at 1000 rpm for 3 min. Cells were resuspended with mTeSR plus with 10 μM Y27632 (Selleckchem, S1049) and evenly distributed on GFR-Matrigel coated wells.

Chen Y., Chauhan S., Gong C., Dayton H., Xu C., De La Cruz E.D., Datta M.S., Leong K.W., Dietrich L.E, & Tomer R. (2023). Scalable projected Light Sheet Microscopy for high-resolution imaging of living and cleared samples. bioRxiv.

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