|Catalog. No.||Product Name||Format||Stock Conc.||Storage on Arrival||Thawing Instructions||Storage Once Thawed|
|ax0664||Human iPSC-Derived Microglia (Male)||Frozen Vial, 1 x 10e6 cells||N/A||-150°C / liquid nitrogen||See Protocol||37°C|
|ax0660||Microglia Maintenance Medium + Supplement A, B and C||100 mL + 100 μL + 100 μL + 1 mL||1x||Store Microglia
Maintenance Medium at
Store supplements A, B and
C at -80°C
|Supplement A – RT
Supplement B – RT
Supplement C – 4°C
|Use immediately and
re-aliquot remaining for
storage at -80°C
Additional Reagents Required
|Product Name||Supplier||Supplier Product Code||Storage on Arrival|
|Thiazovivin||Focus Biomolecules||10-1191 – 5MG||-80°C|
Lot-specific information such as specifications and quality control details are stated in the Certificate of Analysis. Expiry dates for Axol-supplied components are stated on the label. Consult the manufacturer’s guidelines for expiry dates of any additional reagents
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
• Recommended cell culture medium: Microglia Maintenance Medium supplemented with Supplement A, B and C
Preparation of Reagents:
Microglia Maintenance Medium
- Store Supplement A at -80°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°C until required. Do not thaw and refreeze more than once.
- Store Supplement B at -80°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°C until required. Do not thaw and refreeze more than once.
- Store Supplement C 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.
Making up Complete 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|
- Upon receipt, store Thiazovivin vial at -80°C
- Prepare a 10mM stock of Thiazovivin by dissolving 5mg powder in 1606 μL of DMSO.
- Make 50 μL aliquots and store at -80°C for up to 2 months
- Use freshly each time and do not re-freeze
- Upon receipt, store Concanavalin A at -20°C
- Prepare aliquots of Concanavalin A by adding 25 mL sterile DPBS to 25mg of powder for a 1 mg/mL solution.
- Filter the solution through a 0.22um PES filter, make 1 mL aliquots and store at -20°C for up to 3 months.
Coating cell culture plates
Axol’s cryopreserved microglia require both Concanavalin A and Surebond-XF in order to attach to cell culture surfaces. The same coating procedure below can be used for both plastic and glass. Cryopreserved microglia may be thawed directly onto cell culture treated plastic, for example when cells need to be re-plated at a later date. However, this may result in some cell loss and Axol have not performed quality control using this methodology.
- Thaw an aliquot of Concanavalin A
- Add Surebond-XF and Concanavalin A in a 1:100 dilution in sterile DPBS to give a final concentration of 5 μg/mL Surebond-XF and 10 μg/mL Concanavalin A
- Add to culture ware at 1mL per 10cm^2 growth area.
- Incubate coating mixture for at least 4hrs or overnight at 37°C, 5% C0 2
- Aspirate coating and wash thoroughly three times with sterile distilled water in a volume equal to the coating volume
- Leave the culture vessel open in a biological safety cabinet until surface is completely dry
- This can take anywhere between 45 minutes to 3 hours depending on the vessel used
- Coated culture ware should preferably be used the same day, though can be kept overnight at 4 °C without appreciable loss of attachment function
Thawing Human iPSC-derived Microglia
- Prepare Microglia Plating Medium. For this, thaw an aliquot of Thiazovivin and add 1:1000 to Complete Microglia Medium to give a final concentration of 10μM Thiazovivin
- Remove the vial of frozen microglia from liquid nitrogen / freezer storage.
- Ensure the cap is tight. Roll the vial between your gloved hands until the outside is free of frost.
- Immerse the vial in a 37°C water bath until about ¾ of the way down, without submerging the cap. Swirl the vial gently. When only a pea-sized ice crystal remains, remove the vial from the water bath
- Spray the vial with 70% ethanol, wipe dry and bring the vial into the biological safety cabinet
- Transfer the cells gently into a sterile 50 ml conical tube using a 1 ml pipette
- Rinse the vial with 1 ml pre-warmed 37°C Complete Microglia Medium and combine dropwise and slowly with the cell suspension in the 50 ml conical tube, whilst gently moving the tube back and forth. This reduces the effects of osmotic shock and maximizes cell viability.
- Slowly, using a minimal speed setting on an electronic pipette, add 8 ml of warmed, 37°C Complete Microglia Medium to the cells in a drop-wise fashion whilst swirling the tube. This should be done over a 30 second period.
- Centrifuge the cells at 300 x g for 5 minutes.
- Discard most of the supernatant by aspiration and use a P200 pipette to remove the last remnants of supernatant. Be careful not to disturb the pellet
- Re-suspend the cell pellet in 1 ml pre-warmed, 37°C Microglia Plating Medium (Complete Microglia Medium + 10uM Thiazovivin).
- Pipette the cells up and down several times with moderate force to achieve a homogenous single cell suspension
- Take the average of three cell counts to determine the total number of viable cells in the vial
Plating Human iPSC-derived Microglia
The recommended seeding density for Human iPSC-derived Microglia is 100,000 cells/cm 2.
Cells should not be seeded below this density but can be seeded higher is desired.
- Once the total number of viable cells has been determined, dilute the cell suspension in Microglia Plating Medium to obtain the desired plating density
- Mix the cell suspension either by inversion of the tube or with an electronic pipette
- Quickly, add the cell suspension to the coated cell culture vessel and immediately agitate the vessel to disperse the cells evenly across the surface
- Leave undisturbed for 20 minutes on an even surface in a biological safety cabinet to allow the cells to attach
- Incubate at 37°C, 5% C0 2 Human iPSC-derived Microglia can be seeded at densities ranging from 100,000 – 300,000 cells/cm 2
The user however should determine the appropriate seeding density for their application empirically. The table summarises recommended volumes and plating densities for microglia seeded at the recommended 100,000 cells/cm 2
All measures are per well.
|Culture Vessel||Surface Area (cm2)||Plating Volume (mL)||Cell Number to Seed (100K/cm2)||Plating Density (cells per mL)|
|6-well plate||10||2||1 x 106||500,000|
Maintenance of Human iPSC-derived Microglia
• Pre-warm an aliquot of Complete Microglia Medium to 37°C
• The day after seeding, perform a full media change with Complete Microglia Medium
• Perform a half medium change every 2 days thereafter
• Allow cells to recover for at least 7 days before performing assays or experiments