Matrigel (Yeasen, China) was spread evenly on the bottom of a 96-well plate at 50 μl/well and placed in a 37 °C/5 % CO2 incubator for 2 h to allow the Matrigel to solidify. HUVECs were seeded in 96-well plates at 30,000 cells/well, given medium containing PUMSLC-sEVs, and cultured for 4–8 h. Their tube formation was observed under an inverted microscope, and the lengths and branching points of the tubes formed were measured by ImageJ software.
Matrigel
Manufactured by Yeasen
Sourced in China
Matrigel is a soluble basement membrane extract of proteins derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is widely used as an extracellular matrix for the culture of various cell types, including epithelial, endothelial, and stem cells. Matrigel provides a complex mixture of structural and functional proteins that mimic the natural extracellular environment, supporting cell attachment, growth, migration, and differentiation.
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Lab products found in correlation
Te2000, by Nikon (5 mentions)
Crystal violet, by Beyotime (2 mentions)
24 well transwell chamber, by Corning (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
Inverted microscope, by Olympus (1 mentions)
24 well transwell plate, by Corning (1 mentions)
Upper chamber, by Corning (1 mentions)
Matrigel, by Proteintech (1 mentions)
Tgfb1, by Proteintech (1 mentions)
Am6000 microscope, by Leica (1 mentions)
Matrigel, by BD (1 mentions)
Male nude mice, by GemPharmatech (1 mentions)
24 well plate, by Corning (1 mentions)
Optical microscope, by Olympus (1 mentions)
Crystal violet, by Solarbio (1 mentions)
31 protocols using matrigel
1
Angiogenic Potential of PUMSLC-sEVs
2
Transwell Migration Assay for Cell Invasion
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The pre-cooled Matrigel (40111ES08, Shanghai Yeasen Biotechnology, Shanghai, China) diluted with serum-free DMEM at the ratio of 1:8 was used to coat the Transwell apical chamber (3413, Beijing Unique Biotech, Beijing, China) (the step could be ignored in the migration experiment), and it was solidified in a 37°C incubator for 4–5 h. The transfected cells were diluted with 100 μL serum-free medium to prepare a cell suspension at a concentration of about 1 × 106 cells/mL. Later, the cells were inoculated, and 500 μL DMEM containing 20% FBS was added to the basolateral chamber, with 3 replicate wells in each group. The cells were incubated for 24 h at 37°C with 5% CO2. The transwell chamber was washed twice with PBS, fixed with 5% glutaraldehyde at 4°C, stained with 0.1% crystal violet for 5 min, followed by two additional PBS washes. The cells on the surface were wiped with cotton balls. After that, they were analyzed under an inverted fluorescence microscope (TE2000, Nikon Imaging Sales, Shanghai, China). Five fields were randomly selected for photographing (200×), with the mean value selected as the number of cells passing through the chamber in each group. The experiment was repeated three times.
3
Matrigel-based Cell Invasion Assay
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For cell-invasion experiments: Matrigel (YB356234; Shanghai Yubo Biotechnology, Shanghai, PR China) stored at −80°C was removed and liquefied overnight at 4°C. The upper chamber of the chamber bottom membrane was coated with a 1:8 diluent of Matrigel (200 mg/mL) (40111ES08; Yeasen Biotechnology, Shanghai, PR China). The chambers were dried overnight under sterile conditions. The cells were digested, counted, and prepared to suspension with the medium containing 20% FBS. The upper chamber was added with 5 × 104 cells, whereas the lower chamber was added with 800 μL of medium containing 20% FBS and 50 μΜ of VB simultaneously. All cells were incubated in a 37°C incubator for 20−24 h. Transwell plates were rinsed twice with PBS, soaked in formaldehyde for 10 min, and then rinsed three times under running water. The cells were stained with 0.1% crystal violet, allowed to stand for 30 min at room temperature, and rinsed twice with PBS. Afterward, the cells on the surface were wiped off with cotton balls. Five visual fields were randomly selected for photography and documentation under an inverted microscope (XDS-800D; Shanghai Caikon Optical Instrument, Shanghai, PR China).
4
Transwell Assay for Cell Migration and Invasion
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The transwell chamber system (3413, Beijing Youni Biotechnology, China) was used for cell migration and invasion assays. For cell migration assays, A549 cells (1 × 106 cells/mL) in 100 μL serum-free DMEM were added into the upper chambers. For cell invasion assays, A549 cells were added into the upper chambers coated with 50 μL Matrigel (40111ES08, YeaSen Biotechnology, Shanghai, China) pre-diluted (1:1) with serum-free DMEM. After incubation for 24 h, the cells that migrated to the lower chamber containing 500 μL 20% FBS-supplemented DMEM (for cell migration assays) and 600 μL 20% FBS-supplemented DMEM (for cell invasion assays) were subject to fixation with 5% glutaraldehyde and 0.1% crystal violet staining. The stained cells were counted in five microscopic fields (TE2000, Nikon, China) per well, and the average was calculated.
5
Transwell Assay for Cell Migration and Invasion
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The 24-well transwell chambers (8-µm pore size; Corning Costar, Tewksbury, MA, USA) were used to detect cell migration and invasion. A total of 3 × 104 Huh7 cells were resuspended in 200 μL serum-free medium and seeded into the upper chamber pre-coated with (for invasion assay) or without (for migration assay) Matrigel (Yeasen, Shanghai, China). Then, 600 μL complete culture medium was added to the bottom chamber. Plates were maintained in 5% CO2 at 37°C for 24 h. Afterwards, cells that migrated or invaded the lower filter surfaces were fixed with 90% ethanol and stained with 0.1% crystal violet (Beyotime, Shanghai, China). Migrated or invaded cell numbers in five randomly selected fields of view were photographed with an IX81 microscope (Olympus) and quantified using Image-Pro Plus 6.0 software.
6
BALB/c Nude Mice Xenograft Model
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The 4–5 weeks-old female BALB/c nude (nu/nu) mice purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China) were housed under specific pathogen-free (SFP) conditions. Approximately 1 × 107 / mL MDA-MB-231 cells in PBS supplemented with Matrigel (Yeasen, 40183ES10) (1:1) were injected subcutaneously into the BALB/c nude (nu/nu) mice after one week. All animal studies were approved by the Ethics Committee for Animal Experimentation of China Agricultural University.
7
Cell Migration Assay Protocol
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After transfection for 48 hours, cells were starved in serum‐free medium for 24 hours. Then 1E5 cells were resuspended with cell medium containing 10% FBS and added into the transwell chamber coated with diluted Matrigel (Yeason, China). Meanwhile, DMEM cell medium containing 20% FBS was added into basolateral chamber. After cultured for 24 hours, the cells were immobilized using 4% polyformaldehyde then stained with 0.5%‐1% crystal violet solution. The images were captured by microscope (Olympus).
8
Transwell Invasion Assay for Cancer Cells
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SKOV3 and Caov-3 cells were prepared in a suspension of 1 × 105 cells/ml using the serum-free DMEM and then added into the upper part of Transwell chambers coated with 30 μl Matrigel (40111ES08, YeaSen Biotechnology, Shanghai, China) pre-diluted (1:3) with serum-free DMEM. After 24-h incubation at 37°C, the cells that transferred to the lower chamber containing 0.5 ml 10% FBS-supplemented DMEM were fixed in 95% ethanol and stained with 0.1% crystal violet staining, and counted in six microscopic fields per well using an inverted microscope (Olympus, Tokyo, Japan).
9
Pancreatic Tumor Xenograft Implantation
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The pancreas was exposed following the same steps described above. Before injection, the injection areas were warmed using 37°C saline. Next, 3×106 Panc02 cells resuspended in 30μL Matrigel (Yeasen, 40134ES08) were immediately administered into the pancreas capsule. Before needle drawing, a heating lamp was used to heat the injection site to promote solidification of the Matrigel. Finally, the spleen and pancreas were incorporated into the abdominal cavity and the abdomen was closed.
10
Transwell Assay for Cell Migration
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The pre-cooled Matrigel (40111ES08, Shanghai Yeasen Biotechnology Co., Ltd., Shanghai, China) diluted with serum-free DMEM (Matrigel:DMEM = 1:8) was used to coat the Transwell apical chamber (3413, Beijing Unique Biotech Co., Ltd., Beijing, China) (this step was skipped in migration experiments), and was incubated in a 37°C incubator for 4–5 h. The transfected cells were diluted with 100 μl of serum-free medium to prepare a cell suspension, and the concentration was approximately 1 × 106 cells/ml. Next, 500 μl of DMEM containing 20% FBS was added to the basolateral chamber, followed by a 24-h period of incubation at 37°C with 5% CO2. Three duplicate wells were set for each group. The Transwell chamber was then fixed with 5% glutaraldehyde at 4°C and stained with 0.1% crystal violet for 5 min. Then, the surface cells were wiped off using cotton balls. After that, the cells were observed under an inverted fluorescence microscope (TE2000, Nikon Imaging Sales Co., Ltd, Shanghai, China). Five fields (200×) were randomly selected for photographing, and the mean value was taken as the number of cells passing through the chamber in each group.
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