An Assay for Analyzing Test β-Glucan as an Immunotherapy Strategy against Brain Cancer Cells

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.

1. 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.
2. 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).
3. 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.
4. 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.
5. 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

1. 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.
2. 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

1. 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).