Overview
This video demonstrates an in vitro assay utilizing β-glucans to activate microglial cells for the production of superoxide radicals, a reactive oxygen species. In brain cancer cells, the generated reactive oxidants actively suppress tumor cell proliferation and induce cell death.
Protocol
In vitro study of β-glucan-induced microglia stimulation
1. Cell culture of mouse glioblastoma and microglia cells in 8-well chamber slides
NOTE: This protocol is specific for GL261 (glioblastoma) and BV2 (microglia) cell lines. However, with slight modifications, these steps could potentially be used to study other cancer and immune cell lines.
- Prepare Dulbecco's modified Eagle's medium (DMEM) complete medium modified with L-glutamin, 4.5 g/L D-glucose, and without pyruvate. Add 10% of fetal bovine serum (FBS) and 1% penicillin/streptomycin. Pre-warm the material in a water bath at 37 °C for 15 min.
- Thaw frozen BV2 and GL261 aliquots into a water bath (37 °C) for 2 min, and just before they completely thaw, carry them into a laminar flow hood and plate the cells into two different sterile T25 flasks (one for each cell line).
- Incubate the T25 flasks at 37 °C, 5% CO2 until the culture is confluent.
NOTE: Depending on the freezing conditions and the time under cryopreservation, the time until confluence may vary. These cell lines usually require between 3 to 5 days to reach confluency in a T75 flask. - After the BV2 cell culture becomes confluent, transfer it into 8-well chamber slides 0.6 x 106 cells/ well. Keep the 8-well chamber slides in the incubator for 24 h.
- Once the microglia cells are plated into the 8-well chamber slides, repeat the same protocol with the GL261 cells.
2. Activation of microglia with β-glucans
- Coat the BV2 cells with four different β-glucans (P. ostreatus, P. djamor, G. lucidum, and H. erinaceus) at a 0.2 mg/mL concentration for 72 h. One experimental condition must remain untreated (normal medium), acting as the control group.
- Collect the supernatant with a pipette after 72 h and pass the remaining volume through a 0.20 μm syringe filter. Then, freeze the supernatant at -80 °C for at least 24 h.
3. Treatment of GL261 with pre-activated microglia-conditioned medium
- Once the GL261 is 80% confluent within the 8- well chamber slides, add β-glucan-treated microglial medium at a final volume concentration of 25% for 72 h (total volume: 250 μl/well).
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Materials
Name | Company | Catalog Number | Comments |
8-well chamber slides | Thermo Fisher, USA | 171080 | |
Dulbecco's modified Eagle's medium, Gluta MAXTM | Gibco, Life Technologies, Carlsbad, CA, USA | 10564011 | |
Extenda (α- Amylase/Glucoamylase) | Novozymes, Denmark | ||
Fetal bovine serum | Gibco, Life Technologies, Carlsbad, CA, USA | A4736301 | |
Phosphate-buffered saline | Gibco, Life Technologies, Carlsbad, CA, USA | 1010-015 | |
Penicillin/streptomycin | Sigma-Aldrich, St. Louis | P4458 | |
Incubator | Eppedorf | Galaxy 170S |