SB 431542 (hydrate) To achieve a final <0.1% DMSO concentration in the culture medium, dissolve SB431542 in DMSO at highest recommended concentration.
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Dissolve 5 mg SB431542 in 650 μL DMSO for a stock concentration of 20 mM.
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Stock to be aliquoted according to experimental design. For example, if 20 mL of culture media are to be used daily, 10 μL aliquots are prepared and stored at −20°C.
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Use a final concentration of 10 μM in culture medium (1:2,000, 0.5 μL/mL media).
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LDN-193189
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Dissolve 10 mg LDN-193189 in 22.6 mL DMSO for a stock concentration of 1 mM.
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Place tube(s) in water bath (37°C), with occasional vortexing until the compound dissolves, making sure there are no undissolved particles.
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Stock to be aliquoted in volumes suiting the experimental design (for example, if 20 mL of culture media are to be used daily, 2 μL aliquots are prepared and stored at −20°C.
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Use a final concentration of 100 nM in culture medium (1:10,000); dilute once more 1:10 in ddH2O for a more convenient 1:1,000 working stock solution.
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XAV939
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Dissolve 10 mg XAV in 1.6 mL DMSO for a 20 mM stock concentration.
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Aliquot stock and store at −20°C.
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Use a final concentration of 2 μM in culture medium (1:10,000); dilute 1:10 in ddH2O for a more convenient 1:1,000 work stock.
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RHO/ROCK Pathway inhibitor Y-27632
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Dilute to 10 mM in ddH2O
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Small aliquots may be stored at −20°C, avoid repeated freeze-thaw cycles.
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Use a final concentration of 10 μM in culture medium (1:1,000)
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Recombinant human FGF basic
Centrifuge vial containing powder prior to opening. Once dissolved, store at 4°C for 1 week, or −20°C to −80°C for long-term storage. Avoid freeze/thaw cycles; recommended storage time in this form and temperature is approximately 12 months.
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Reconstitute in 1× PBS 0.1% BSA to a bFGF concentration of 10 μg/mL. Do not vortex.
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Aliquot stock solution: e.g., 25 μL into 25 mL of culture medium (if used at 1:1,000).
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Use a final concentration of 10 ng/mL in culture medium.
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Recombinant human EGF basic
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Once dissolved, store at 4°C for 1 week, or −20°C to −80°C for long-term storage. Dissolve 1,000 μg in 10 mL of 1× PBS (0.1% BSA added for long-term storage). This results in a 100 μg/mL solution (10,000× stock)
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Aliquot 10 mL volume into suitable smaller volumes, for example 100 μL, and store at −20°C.
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Use a master/stock aliquot to make a 10× dilution and prepare working solution aliquots (for example, use 100 μL and add 900 μL of 1× PBS (0.1% BSA) to make a 10× 100 μL aliquot 1:1000 working solution.
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Use final concentration of 10 ng/mL in culture medium.
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Poly-L-ornithine hydrobromide
Prepare in cell culture grade water by dissolving powder to a 10 mg/mL solution, dilute to 0.1 mg/mL with ddH2O, and further dilute 1:500 for the stock solution.
The stock solution can be stored at 4°C for several weeks, and at −20°C for long-term storage. |
Geltrex basement membrane matrix
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Thaw stock vial in a beaker containing ice, in a 4°C refrigerator overnight (12–16 h).
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Keep tubes on ice while making aliquots.
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Ready to use Geltrex working solution is prepared by adding the 250 μL stock in a 50 mL Eppendorf tube containing 24.75 mL of Knockout DMEM. Aliquot 250 μL of working solution in 0.5 mL tubes.
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For coating, a stock solution aliquot is thawed on ice or at 4°C and diluted 1:100 in serum-free media (see above).
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Coat using a volume that will cover the surface of the well/plate (e.g., 1 mL for a well in a 6-well plate); tilt culture plate sideways till surface is covered, making sure there are no dry patches.
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Place culture plate in incubator (37°C) for 1 h, before use.
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Remove Geltrex solution and seed cell suspension.
Precoating: for long-term storage of coated plates, seal plates with parafilm and store at 4°C for a maximum of 2 weeks. Before use, check that surface is covered and that Geltrex has not solidified or dried out. |
Fibronectin
Dissolve contents of vial in cell culture grade water. Dilute to 1 mg/mL in ddH2O by gently warming the vial to 37°C. This is further used as a 1:1,000 stock solution.
Do not agitate as it may cause precipitation.
Store stock solution at −20°C. |
N2 and B-27 thawing and aliquoting
See manufacturer’s instructions for N2 and B-27 supplements. |
