UC-MSCs from passage 4 were used for IPC differentiation (Figure 1 ). Undifferentiated cells were suspended in complete culture media and aliquots of 2.5 × 105 cells, and placed in 6-well plates overnight. The medium was replaced with complete culture media either with or without pre-coated 5 μg/mL laminin 411 (Biolamina, Stockholm, Sweden) and 25 mM glucose for 3 d (stage I). At stage II, the media were refreshed with DMEM/F-12 medium containing Insulin-Transferin-Selenium-A (ITS-A, BD) for another 4 d. At stage III, 10 mM nicotinamide (MP, USA) was added into the media, and the culture lasted for 3 days. At stage IV, the medium was changed with the same supplements as at stage III, but N2 and B27 supplements (Invitrogen) were added and incubated for 4 d.
Laminin 411
Manufactured by BioLamina
Sourced in Sweden, Germany
Laminin-411 is a protein that is a component of the extracellular matrix. It is a key structural element of the basement membrane and plays a role in cell adhesion and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Merck Group (2 mentions)
Rpmi 1640, by PAN Biotech (2 mentions)
N2 and b27 supplements, by Thermo Fisher Scientific (1 mentions)
Celltracker green cmfda dye, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Recombinant human vegf, by Thermo Fisher Scientific (1 mentions)
Stempro 34 sfm, by Thermo Fisher Scientific (1 mentions)
Iwr 1, by Merck Group (1 mentions)
5 protocols using laminin 411
1
Directed Differentiation of UC-MSCs to IPCs
2
Preparation and Storage of Archazolid A
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Archazolid A (hereinafter referred to as archazolid) was kindly provided by Prof. Dr. Rolf Müller (Saarland University) and Prof. Dr. Dirk Menche (University of Bonn). The compound was dissolved in dimethylsulfoxide (DMSO). Stocks of 10 mM archazolid were stored at -80°C and working stocks of 10 μM were stored at -20°C. For the treatment of cells archazolid was diluted in culture medium with a maximal concentration of 0.01% DMSO. Collagen G and fetal calf serum (FCS) were purchased from Biochrom (Berlin, Germany), recombinant tumor necrosis factor α (TNFα) from PeproTech (Hamburg, Germany), bovine serum albumin (BSA) from Carl Roth (Karlsruhe, Germany), CellTracker Green CMFDA Dye from Thermo Fisher Scientific (Schwerte, Germany), human plasma fibronectin (FC010) from Merck (Darmstadt, Germany) and laminin-411 from BioLamina (Sundbyberg, Sweden).
3
MCAM and Laminin-411 Coculture Protocol
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Cell culture plates were coated with human recombinant MCAM (Thermo; 10 µg/ml)/laminin-411 (Biolamina; 10 µg/ml) for 2 h at RT. The cells were diluted to 1*10^6/ml in RPMI1640 (PAN Biotech) + 10% FCS (Sigma) + 1% PS (PAN Biotech) and incubated rolling on the coated plates on a shaker for 1 h at RT at 100 rpm, harvested in 100 µl Rap/Rac lysis buffer (50 mM Tris PH = 7.4, 500 mM NaCl, 25 mM NaF, 2.5 mM MgCl2, 1 mM NaOrthovanadate, 10% glycerol, 1% NP40; all Sigma) complemented with HALT protease cocktail (1:100; Thermo).
4
Immunofluorescence Staining of Adherent Cells
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Glass cover slips were coated with human recombinant MCAM (Thermo; 10 µg/ml)/laminin-411 (Biolamina; 10 µg/ml) for 2h at RT. The cells were diluted to 1*10^6/ml in RPMI1640 (PAN Biotech) + 10% FCS (Sigma) + 1% PS (PAN Biotech) and incubated rolling on the coated cover slips on a shaker for 1h at RT at 100 rpm. Then, 8% PFA (Sigma) in PBS was carefully added (final concentration 4% PFA) to the cells to fix them in situ. After incubating at RT for 10 min, the cells were permeabilized in PBS (PAN Biotech) + 100 mM Glycine (Sigma) + 0.1% triton (AppliChem) for 10 min at RT. Cells were stained in Alexa Fluor647 Phalloidin (Thermo; 1:40 in PBS + 1% BSA) for 30 min at RT in the dark. Next, the cells were carefully washed in PBS and stained with DAPI (Sigma; 1µg/ml in PBS) for 10 min at RT in the dark. Cells were washed in PBS and mounted on object slides using fluorescence mounting medium (Dako).
5
Differentiation of LPM and Vascular Cells
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For differentiation toward LPM and vascular cells, CDX1 + cells were cultured with 3 mM CHIR99021, 100 ng/ml bFGF and 25 ng/ml BMP7 for 3 days. Then, the cells were dissociated with Accutase and replaced on Laminin 411 (BioLamina)-coated 24-well plates in StemPro-34 SFM (Thermo Fisher Scientific) supplemented with 50 ng/ml recombinant human VEGF (Peprotech), 10 ng/ml bFGF, 10 mM DAPT (R&D Systems), 20 ng/ml BMP4 and 10 mM IWR1 (SIGMA) for 4 days.
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