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Instruction manual for culturing Axol iPSC-derived microglia (96-well plate format)

Product Information

Catalog. No. Product Name Format Stock Conc. Storage on Arrival Thawing Instructions Storage Once Thawe
ax0666 Plated Human iPSC-Derived Microglia (Female) 1x 96-well plate 30,000 cells/well N/A 37ºC N/A N/A
ax0660 Microglia Maintenance Medium + Supplement A, B and C 100 mL + 100 μL + 100 μL + 1 mL 1x Store Microglia Maintenance Medium at 4ºC Store supplements A, B and C at -80ºC Supplement A – RT Supplement B – RT Supplement C – 4ºC Use immediately and re-aliquot remaining for storage at -80ºC

Lotspecific information such as specifications and quality control details are stated in the Certificate of Analysis. Expiry dates for components are stated on the label.

Important! Axol Neural Cell Culture Media
DOES NOT contain antibiotics or antifungal agents. Axol 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

Preparation of Reagents

Supplement A

  • Store Supplement A at -80 o C on arrival.
  • Thaw at room temperature and use 50 μL of Supplement A to make up 50 mL of Complete Microglia Maintenance Medium. Aliquot the remaining 50 μL and store at -80 o C until required. Do not thaw and refreeze more than once.

Supplement B

  • Store Supplement B at -80 o C on arrival.
  • Thaw at room temperature and, use 50 μL of Supplement B to make up 50 mL of Complete Microglia Maintenance Medium. Aliquot the remaining 50 μL and store at -80 o C until required. Do not thaw and refreeze more than once.

Supplement C

  • Store Supplement B at -80 °C on arrival.
  • Thaw at 4 ° C. Use 500 μL of Supplement C to make up 50 mL of Complete Microglia Maintenance Medium. Aliquot the remaining 500 μL and store at -80°C until required. Do not thaw and refreeze more than once.

Microglia Maintenance Medium

  • Upon receipt, aliquot and store Microglia Maintenance Medium at 4 °C.
  • Microglia Maintenance Medium requires supplementing with Supplement A, B and C before use.
  • Add the Supplements to the Microglia Maintenance Medium according to the table.
  • Complete, fully supplemented, Microglia Maintenance Medium is stable for 1 week at 4°C.
  • Before use, pre-warm an aliquot of Microglia Maintenance Medium to 37ºC.
Supplement Final Concentration 50 mL Microglia Maintenance Medium
Supplement A 10 ng/mL 50 μL
Supplement B 100 ng/mL 50 μL
Supplement C 1 x 500 μL

Maintenance of Human iPSC-Derived Microglia

Axol’s Human iPSC-Derived Microglia are derived from iPSCs which have undergone an intermediate differentiation to microglial precursors prior to induction of terminal differentiation.

The 96-well plate will arrive in a sealed plastic bag and the wells will be capped tightly with a silicone seal. For worldwide shipments (non-European), Plated Human iPSC-Derived Microglia will be shipped on Day 7 of differentiation from precursors to arrive on Day 9 or Day 10. Upon arrival of the cells, perform a half medium change, then every other day thereafter.

Unless otherwise specified by the customer, UK and European shipments of Plated Human iPSC-Derived Microglia are normally sent on Day 14 of differentiation to arrive the following day. UK and European customers should allow a 24-hour recovery period after receipt of the cells before
conducting assays or other experiments. Otherwise, perform a half medium change the day after receipt of the cells (Day 16), then every other day thereafter.

Consult the feeding schedule on page 6 for more information.

  • Carefully open the plastic packaging without knocking or dropping the plate. Use of scissors is recommended. Place the 96-well plate into a class II biosafety cabinet, making sure to spray the plate with 70% ethanol and wipe it down first.
  • Remove the silicone sealing cap from the plate.
  • Check cells under the microscope.
  • Incubate the plate at 37 °C, 5% CO2 .
  • On the day of receipt (worldwide shipments) or day after receipt of plate (UK/European) shipments), gently replace 100 μL of medium with 100 μL of fresh, pre-warmed 37 °C, Microglia Maintenance Medium.
  • Incubate the plate at 37 °C, 5% CO2 .
  • Perform a half medium change every other day
  • Human iPSC-Derived Microglia must be cultured to at least Day 14 at which point they become fully mature and are ready to use for experiments. Optimally, preferred assays should be conducted between Day 14 and Day 21
  • Axol does not recommend maintaining the cells past Day 21 as after this point the quality of the culture begins to deteriorate
  • Below is an example of a healthy microglia culture at Day 10 after a 3-day transatlantic shipment

UK/European Feeding Schedule

Worldwide Feeding Schedule

If the plate arrives on Day 9, feed on Days 9, 11, 13, 15, 17, 19, and 21
If the plate arrive on Day 10, feed on Days 10, 12, 14, 16, 18, and 20