Tet-mCherry or Tet-MyoD hiPSCs were cultured on Easy iMatrix-511 silk-coated plates (#892024, Nippi) in StemFit medium (AK02N, Ajinomoto) containing 100 μg/mL G418 (#938044, NacalaiTesque) or 0.5 μg/mL puromycin dihydrochloride (160-23151, Wako Chemicals). Cells were passaged every 7 days using Accutase (#12679-54, NacalaiTesque) and seeded on Easy iMatrix-511 silk-coated 6-well plates in the presence of 10 μM Y-27632 (NacalaiTesque) at a density of 1.5×104 cells/well for the first 2 days after plating. At 48 h after passaging, Y-27632 was removed and replaced with StemFit medium containing the appropriate antibiotic.
Easy imatrix 511 silk
Manufactured by Nippi
Sourced in Japan
Easy iMatrix-511 silk is a cell culture matrix derived from recombinant laminin-511 protein. It provides a defined, animal-component-free substrate for the culture of human pluripotent stem cells and other cell types.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (2 mentions)
Puromycin dihydrochloride, by Fujifilm (1 mentions)
Accutase, by Nacalai Tesque (1 mentions)
Y 27632, by Nacalai Tesque (1 mentions)
Stemfit medium, by Ajinomoto (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Ak02n, by Nippi (1 mentions)
Rock inhibitor, by Nacalai Tesque (1 mentions)
Rpmi 1640 medium, by Nacalai Tesque (1 mentions)
Stemfit ak02n, by ReproCELL (1 mentions)
5 protocols using easy imatrix 511 silk
1
Maintenance of Tet-inducible hiPSC Lines
2
Coating Matrices for Cell Culture
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For laminin-coated plates, 1.5 mL or 8 mL of Easy iMatrix-511 silk (Nippi) was added to each well of the 6-well plates or 10 cm dishes, after which the plates were incubated overnight at 4°C. Coated plates were stored at 4°C for up to 2 weeks until use. Laminin-coated plates were equilibrated at 24°C for at least 30 min before use in the experiments. For Matrigel-coated plates, all tubes, pipette tips, and reagents were pre-chilled in the refrigerator or on ice. Matrigel (BD Falcon, Franklin Lakes, NJ, USA) was prepared at a 1:100 dilution with pre-chilled media without any additives. An appropriate volume of diluted Matrigel was dispensed into each well of the 6-well or 96-well plates. Plates were incubated at 4°C overnight before use. Matrigel-coated plates were stored at 4°C for up to several weeks until use. For collagen gel plates, 1.2 mL or 10 μL of 0.5 mg/mL collagen solution (Nippi) was dispensed into a well of 6-well (BD Falcon) or a μPlate Angiogenesis 96 plate (Ibidi, Planegg, Germany), and a layer of 1-2 mm collagen gel was solidified in an incubator at 37°C overnight. Next day, collagen gel was further coated with Matrigel at 4°C overnight before the plating of the cells.
3
Assessing Epithelial Barrier Function
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HTS Transwell clear 12-well plates were purchased from Corning, and the inner chambers, which contained polycarbonate membrane 12 mm in diameter with 0.4 μm pores, were coated with Easy iMatrix-511 silk (Nippi; 0.25 μg/cm2; preincubated for 1 h at 37 °C). P4 dUCs or aHDFs were resuspended in UCM and seeded in the inner chamber at a density of 1 × 105 cells/well. HUCs were resuspended in Urolife D Complete Medium (Urolife) and seeded in the inner chamber at a density of 1 × 105 cells/well. For terminal differentiation, some aliquots of HUCs were cultured in Urolife D Complete Medium supplemented with 1 μM Rosiglitazone and 1 μM PD153035 for 4 days48 (link). Cells were cultured for 5 days followed by washing with PBS(-). FITC-Dextran with an average molecular weight of 4 kDa (IWAI Chemicals) was added to the inner chamber at a concentration of 2 mg/mL. After incubation for 20 min in the dark, the supernatant in the outer chamber was transferred into 96-well plates, and fluorescence intensity in each well was measured by SpectraMax M2 (Molecular Devices) with excitation and emission wave lengths at 535 nm and 485 nm, respectively. Percentage of leaked FITC-dextran was calculated as follows: (Fluorescent count of each well)/(Fluorescent count of no cell control well) × 100.
4
Culturing Cell Lines for Research
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aHDFs and Plat-GP cells were purchased from ScienCell Research Laboratories and Cell Biolabs, respectively, and cultured in Standard Medium. aHDFs at passage number less than 10 were used for experiments. HUCs derived from a 24-years-old male African American were purchased from KURABO Industries (KP-4309; Lot 04101). Induced pluripotent stem (iPS) cells were purchased from JCRB Cell Bank and maintained as previously described43 . Briefly, iPS cells were cultured in StemFit medium (Ajinomoto; AK02N) on Easy iMatrix-511 silk (Nippi; 0.25 μg/cm2; preincubated for 1 h at 37 °C)-coated plates with. For passage, iPS cells were dissociated into a single cell suspension in StemFit medium with Rock inhibitor (Nacalai Tesque; Y-27632) and reseeded in new culture plates. On the next day, medium was replaced by fresh StemFit medium without Rock inhibitor, followed by medium change every other day. Passage was performed when iPS cells reached 80% to 90% confluency. T24, human bladder carcinoma cell line, was a kind gift from professor Ashihara (Kyoto Pharmaceutical University, Kyoto, Japan), and cultured in RPMI1640 medium (Nacalai Tesque) supplemented with 10% FBS, MEM nonessential amino acids, 100 mM sodium pyruvate and 100 U/mL penicillin-streptomycin.
5
Extracellular Matrix Coating Protocols
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For the preparation of laminin-coated plates, 1.5 or 8 mL of Easy iMatrix-511 silk (Nippi; Tokyo, Japan) were added to each well of the six-well plates or a 10-cm dish, respectively, and incubated overnight at 4 °C. The coated plates were stored at 4 °C for up to 2 weeks and equilibrated at 24 °C for at least 30 min prior to use. StemFit AK02N (StemFit; Reprocell; Beltsville, MD, USA) (750 µL or 3 mL) was added to wash the plates and aspirated. For the Matrigel-coated plates, all tubes, pipette tips, and reagents were pre-chilled in the fridge or on ice. Matrigel (BD; Franklin Lakes, NJ, USA) was prepared at 1:100 dilution with pre-chilled media without any additives. An appropriate volume of diluted Matrigel was dispensed into each well of the 6- or 96-well plates, which were incubated overnight at 4 °C and stored at 4 °C for a few weeks until use. For the gel culture, 2 mL or 10 µL of 0.5 mg/mL collagen solution (Nippi) were dispensed into a well of 6-well plates (BD) or µPlate Angiogenesis 96 plate (Ibidi; Planegg, Germany) and solidified in an incubator at 37 °C overnight. The plates were then coated with Matrigel.
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