FACS-sorted cells were resuspended at a density of 4 × 105 cells per milliliter in chilled 100% growth factor-reduced Matrigel (BD Bioscience), and the mixture was allowed to polymerize before covering with culture medium (DMEM/F12; ITS [1:100; Sigma]; 50 ng mL−1 EGF; plus either vehicle [1% CHAPS in PBS], 200 ng mL−1 Wnt3A [Willert et al. 2003 (link)], 200 ng mL−1 Dkk1, 1 μM E2, 2.5 μM Pg, or 2uM IWP2). Culture medium was changed every 24 h. Primary colony numbers were scored after 6–7 d in culture. The colonies were mostly spherical. In cases in which colonies were oval, the long axis was measured. For passaging colonies, the medium was aspirated, and Matrigel was digested by incubation in 500 μL of Matrigel recovery solution (BD Bioscience) for 1 h on ice. Colonies released from Matrigel were harvested after pelleting. Single cells were obtained through incubation with 0.25% Trypsin-EDTA for 5 min at 37°C followed by gentle pipetting. Single cells were then replated in Matrigel as described above.
Matrigel recovery solution
Manufactured by BD
Matrigel recovery solution is a specialized liquid designed for the extraction and collection of Matrigel, a complex extracellular matrix derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. This solution facilitates the recovery of Matrigel from various laboratory surfaces and equipment.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (5 mentions)
Dmem f12 its, by Merck Group (2 mentions)
Lithium chloride (licl), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Rabbit anti sox9, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Rhodamine conjugated phalloidin, by Merck Group (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
Mouse anti ki67, by BD (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Rabbit anti phospho histone h3 ph3, by Merck Group (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Dmem f12k medium, by Thermo Fisher Scientific (1 mentions)
Jagged 1, by AnaSpec (1 mentions)
6 protocols using matrigel recovery solution
1
3D Culture of Mammary Stem Cells
2
3D Organoid Culture of Stem Cells
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FACS-sorted cells were infected with lentivirus overnight, and resuspended at a density of 4 × 105 cells ml−1 in chilled 100% growth-factor-reduced Matrigel (BD Bioscience). The mixture was allowed to polymerize before being covered with culture medium [DMEM/F12, ITS (1:100; Sigma)] which was changed every 24 h. Primary colony numbers were counted and the diameters were measured after 5–7 days in culture. The colonies were mostly spherical, if colony was oval, the long axis was measured. LiCl (Sigma) was added to the culture medium from day 3. For Wnt treatment, 200 ng ml−1 Wnt3A or Wnt4 conditional medium (1:50 conditional medium from Wnt4-expressing EpH4 stable cell line) was added from day 1. For passaging colonies, the medium was aspirated, and Matrigel was digested by incubation in 500 μl of Matrigel recovery solution (BD Bioscience) for 1 h on ice. Colonies released from Matrigel were harvested after pelleting. Single cells were obtained through incubation in 0.25% Trypsin-EDTA for 5 min at 37°C followed by gentle pipetting. Single cells were then replated in Matrigel as described above.
3
Immunofluorescence Analysis of Lung Explant Cultures
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Lung explant cultures were fixed in MEMFA (3.8% formaldehyde, 0.15 M MOPS, 2 mM EGTA, 1 mM MgSO4, pH 7.4) and permeabilised with 1% Triton-X 100 (Sigma-Aldrich, Poole, UK). Isolated epithelial cultures were first removed from culture using Matrigel recovery solution (BD Bioscience) before being fixed with 4% paraformaldehyde and permeabilised with 0.05% saponin.
Samples were then blocked with 2% BSA in PBS and incubated with either mouse anti-E-Cadherin (Cat No: 610182; BD Bioscience), rabbit anti-SOX9 (Cat No: AB5535; Millipore, Watford, UK), mouse anti-Ki67 (Cat No: 610968; BD Bioscience), rabbit anti-phospho-histone H3 (PH 3; Cat No: 05-806; Millipore), rabbit anti-TTF1 (Nkx2.1; Cat No: WRAB-1231; Seven Hills, Cincinnati, USA) or rabbit anti-pro-surfactant protein C (SPC; Cat No: WRAB-9337; Seven Hills). Samples were then incubated with either FITC conjugated horse anti-mouse (Vector labs, Birmingham, UK) or Alexa Flour-568 donkey anti-Rabbit (Invitrogen, Paisley, UK) secondary antibodies prior to mounting. Where indicated, Nuclei and F-actin were visualised by incubating the samples with DAPI and rhodamine-conjugated phalloidin (Sigma-Aldrich) respectively prior to mounting.
Fluorescent images were acquired using a Zeiss LSM 510 confocal microscope.
Samples were then blocked with 2% BSA in PBS and incubated with either mouse anti-E-Cadherin (Cat No: 610182; BD Bioscience), rabbit anti-SOX9 (Cat No: AB5535; Millipore, Watford, UK), mouse anti-Ki67 (Cat No: 610968; BD Bioscience), rabbit anti-phospho-histone H3 (PH 3; Cat No: 05-806; Millipore), rabbit anti-TTF1 (Nkx2.1; Cat No: WRAB-1231; Seven Hills, Cincinnati, USA) or rabbit anti-pro-surfactant protein C (SPC; Cat No: WRAB-9337; Seven Hills). Samples were then incubated with either FITC conjugated horse anti-mouse (Vector labs, Birmingham, UK) or Alexa Flour-568 donkey anti-Rabbit (Invitrogen, Paisley, UK) secondary antibodies prior to mounting. Where indicated, Nuclei and F-actin were visualised by incubating the samples with DAPI and rhodamine-conjugated phalloidin (Sigma-Aldrich) respectively prior to mounting.
Fluorescent images were acquired using a Zeiss LSM 510 confocal microscope.
4
Matrigel Culture and Passaging of Stem Cells
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FACS-sorted cells were resuspended at a density of 4 × 105 cells per milliliter in chilled 100% growth factor-reduced Matrigel (BD Bioscience), and the mixture was allowed to polymerize before covering with culture medium [DMEM/F12; ITS (1:100; Sigma); 50 ng/ml EGF; and either DMSO or 100 μM baicalin]. Culture medium was changed every 24 h. Primary colony numbers were scored after 6–7 days in culture. For passaging colonies, the medium was aspirated, and Matrigel was digested by incubation in 500 μl of Matrigel recovery solution (BD Bioscience) for 1 h on ice. Colonies released from Matrigel were harvested after centrifuging. Cells were obtained through incubation with 0.25% Trypsin-EDTA for 5 min at 37°C followed by gentle pipetting. Cells were then replated in Matrigel as described above. In assays examining the requirement for baicalin in maintaining stem cell properties, baicalin was withdrawn for 5 days in secondary culture prior to transplantation.
5
3D Stem Cell Culture of MDA-MB-231 Cells
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Human triple-negative breast cancer cell line MDA-MB-231 was purchased from ATCC, and cultured in DEME/F12K medium with 10% fetal bovine serum and in aired with 5% CO2 at 37 °C. The 3D stem cell culture was based on a semisolid MatrigelTM system according to a previous publication (Bahmad et al., 2018 ). In brief, single cell suspensions that obtained from cultured MDA-MB-231 cells were resuspended in a 1:1 mixture of Matrigel (BD Biosciences), DMEM/F12K medium containing B27 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), epidermal growth factor (EGF, 20 ng/ml; Solarbio Science and Technology Co., Ltd., Beijing, China), and basic fibroblast growth factor (bFGF20 ng/ml, Solarbio Science and Technology Co., Ltd., Beijing, China), before being plated in 6-well plates at a density of 5000 cells/well. Cells were then cultured with medium as described for 14 days, and the medium was changed every 3 days. After that, the cultured spheres were retrieved using Matrigel Recovery Solution (BD Biosciences) as per the manufacturer’s protocol. The stemness was detected by flow cytometry after staining with CD24, CD44, and CD133. The 2D cultured MDA-MB-231 cells as described were used for control.
6
Organoid Formation from FACS-Sorted Cells
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