Human ipsc-derived Microglial Precursors

Transcription

1 Human ipsc-derived Microglial Precursors

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3 Product Information Catalog. No. Product Name Format Storage on Arrival ax1666 Human ipsc-derived Microglial Precursors 3 x 10 6 viable cells Transfer cells to a 37 o C CO2 incubator as per instruction below ax0660 Microglia Maintenance Medium Kit (Includes Basal Medium + Supplement A, B and C) 100 ml (Basal) / 100 µl (Supp. A) µl (Supp. B) + 1 ml (Supp. C) Store Microglia Maintenance Medium (Basal) at 4 o C for up to 2 months Store supplements A, B and C at -80 o C for up to 1 year Batch-specific product information such as QC test data are stated in the Certificate of Analysis. Important! Axol Cell Culture Media DOES NOT contain antibiotics or antifungal agents. Axol Bioscience does not recommend the use of antimicrobial agents such as penicillin, streptomycin and amphotericin. Antimicrobial agents should not be necessary if proper aseptic technique is adopted. Recommendations Recommended cell culture medium: Microglia Maintenance Medium supplemented with Supplement A, B and C

4 Human ipsc-derived Microglial Precursors Preparation of reagents Supplement A, B & C Store Supplement A, B & C at -80 o C on arrival. After initial thaw, use a portion of each according to the ratios displayed in the table below to make up a desired volume of Microglia Maintenance Medium. Aliquot the remaining amount and store at -80 o C until required. Do not thaw and refreeze more than once. Microglia Maintenance Medium Upon receipt, store Microglia Maintenance Basal Medium at 4 o C. Microglia Maintenance Medium requires supplementing with Supplement A, B and C before use. Add the Supplements to the Microglia Maintenance Basal Medium according to the ratios displayed in the table. Once Supplements A, B and C have been added to the Microglia Maintenance Medium, it is stable for 1 week at 4 o C. Decisions about aliquot volumes of Supplements A, B and C should be made on the basis of this stability information and the calculated rate of medium usage over the differentiation period. Component Product Code Stock Concentration Final Concentration For 50 ml Microglia Maintenance Medium Microglia Maintenance Medium (Basal) ax0660d 1X 1X 49.5 ml Supplement A ax0660a 10 µg/ml 10 ng/ml 50 µl Supplement B ax0660b 100 µg/ml 100 ng/ml 50 µl Supplement C ax0660c 100X 1X 500 µl Before use, pre-warm an aliquot of Microglia Maintenance Medium at 37ºC. Axol Bioscience 4

5 Human ipsc-derived Microglia Seeding of Human ipsc-derived Microglial Precursors Axol s Human ipsc-derived Microglial Precursors are derived from ipscs which have undergone differentiation to monocytes. The cells should be cultured for at least 14 days before use and then used for experiments within 7 days. Human ipsc-derived Microglia Precursors will arrive warm at 37 o C in a cryovial. Gently pipette the microglia to get them into suspension and transfer the cells to a 15 ml conical tube (~ 2.4 ml). Wash the vial twice with 1 ml room temperature Microglia Maintenance Medium (basal, non-supplemented) each time Add a further 7.6 ml room temperature Microglia Maintenance Medium to the 15 ml conical tube to make up to 10 ml. Centrifuge the cells at 300 x g for 5 minutes. Remove and discard the supernatant. Resuspend the cell pellet in room temperature Microglia Maintenance Medium. Perform a cell count to determine total number of viable microglia precursors. ipsc-derived Microglial Precursors in Microglia Maintenance Medium can be seeded directly onto tissue culture-treated plasticware. Axol recommends seeding at a density of 100,000 cells/cm 2. Three million cells (1 vial) are enough for seeding 1x 96-well plate. Incubate at 37 o C, 5% CO2 Terminal Differentiation to Mature ipsc-derived Microglia Perform a half media change with Microglia Maintenance Medium on Days 2 and 3, then every other day thereafter. 5

6 Human ipsc-derived Microglia Representative images of differentiating ipsc-derived microglial precursors Differentiated ipsc-derived microglia at day 18 post plating (Assay time window: day 14-21) 6

7 Human ipsc-derived Microglia Got any questions? Need help with the protocol? Contact Axol Technical Support at Or Call +44 (0)