Fungal beta-glucans are important modulators of the immune system. Enhancing the purity and isolation protocols is essential to asserting their potential role as co-adjuvants for cancer therapy. A high degree of four different beta-glucans was achieved using various devices commonly found in molecular biology and proteomic laboratories.
This protocol focuses on testing the effects of beta-glucans on microglia, the brain's most abundant resident immune cell. Modulating the microglia phenotype in glioblastoma can yield in important insights into. Begin the extraction of soluble mushroom polysaccharides or SMPs by rinsing fresh fruiting bodies of Pleurotus ostreatus, Pleurotus djamor, Hericium erinaceus, and Ganoderma lucidum with distilled water five times.
Then dry the washed fruiting bodies completely at 50 degrees Celsius in a conventional air drying oven for approximately 24 hours before grinding them in a blade mill obtaining about 200 grams of powder from each mushroom. Next, suspend 100 grams of all four mushroom powders or MP in 1, 000 milliliters of distilled water and autoclave the suspension at 121 degrees Celsius for 15 minutes. After 30 minutes of incubation at room temperature, centrifuge the resulting suspension at 6, 000 G for 10 minutes at four degrees Celsius.
Then dry the precipitate containing insoluble mushroom polysaccharides or IMPs at 50 degrees Celsius in an air drying oven for 24 hours. Discard the precipitate and concentrate the supernatant 10 times in a rotary evaporator. Next, precipitate the concentrate containing SMPs with ethanol overnight at four degrees Celsius.
After centrifuging the ethanol suspension at 6, 000 G for 15 minutes at four degrees Celsius, discard the supernatant with a pipette and wash the pellet three times with 80%ethanol before dissolving it and distilled water. Adjust the pH to 6.5 to 7 and the temperature to 37 degrees Celsius and solubilize the alpha-glucans by treating the suspension with two units and four units of alpha-amylase and glucoamylase respectively. Next, adjust the pH to 8.0 and temperature to 50 degrees Celsius and solubilize the proteins by treating the suspension with alkalase equivalent to 2.5 units per gram of protein.
After hydrolysis, centrifuge the hydrolysate and clean the supernatant concentrate five times in a rotary evaporator. Precipitate the supernatant concentrate again by adding 80%ethanol. To remove low molecular weight molecules, solubilize the precipitate in distilled water and dialyze it in the distilled water for 24 hours using 12, 000 Dalton cutoff cellulose tubing membranes.
Recover the water soluble portion and freeze dry it to produce soluble beta-glucans or S beta-Gs. Evaluate the total glucans and alpha-glucans separately. Measure the resulting beta-glucan values as the difference between the total glucan and alpha-glucan values.
To obtain the S beta-G UV spectra using a UV visible spectrophotometer, prepare 1.0 milligram per milliliter of each S beta-G in distilled water. Transfer the sample to a quartz cuvette and scan at room temperature in the 200 to 400 nanometers region. Estimate the homogeneity of S beta-Gs and molecular weight of polymers by size exclusion chromatography or SEC using a high performance liquid chromatography or HPLC system equipped with a UV visible detector and a Superdex TM 200 10/300 GL gel filtration column.
Record the infrared spectra of the sample on a Fourier-transform infrared or FTIR spectrometer in the 4, 000 to 500 per centimeter range. Activate the microglia by coating the BV2 microglia cells for 72 hours with 0.2 milligrams per milliliter concentration of four different beta-glucans isolated from P.ostreatus, P.djamor, G.lucidum, and H.erinaceus. After 72 hours, collect the supernatant with a pipette and pass the remaining volume through a 0.20 micrometer syringe filter.
Then freeze the supernatant at minus 80 degrees Celsius for at least 24 hours. To treat GL 261 with the pre-activated microglia conditioned medium, add 250 microliters of beta-glucan-treated microglial medium to 80%confluent GL 261 cells for 72 hours. Discard the medium after 72 hours.
UV spectra of the different S beta-Gs did not show UV absorption peaks at 260 and 280 nanometers, indicating that S beta-Gs lacked nucleic acids and proteins. The HPLC chromatograms showed good homogeneity with a main sharp and single peak at 10.9 minutes for P.ostreatus, 10.5 minutes for lucidum, 11.3 minutes for P.djamor, and 8.20 minutes for H.erinaceus, suggesting the fraction is consistent with homopolymers. The FTIR spectra measured the molecular vibrations corresponding to covalent polysaccharide bonds.
It showed that SS beta-Gs comprised carbohydrates conjugated with a minimal protein. HPTLC and GC-MS analyzing the monosaccharide profile of S beta-Gs revealed the presence of a large amount of D-glucose, smaller amounts of D-galactose and D-mannose, and a trace of D-xylose, D-rhamnose, D-fucose, and L-arabinose. Using an in-house script in ImageJ software, the number of positive pixels for each fluorescent channel was quantified.
The script used control samples as a threshold for the intensity of each fluorophore and provided the number of pixels. It indicated the expression for each marker after different experimental conditions. Interestingly, GL 261 did not suffer significant differences regarding tumor proliferation on exposure to the different microglia conditioned media.
However, P.djamor and H.erinaceus showed strong induction of an approximately sixfold and ninefold increase in cleaved caspase-3 respectively. Ensuring a high-grade purity of beta-glucans is essential for this protocol. This protocol uses advanced equipment such as spectrophotometer, HPLC.
or FITR. Using these tools enhance the robustness of our findings. We can confidently analyze the effects of beta-glucans on microglia and obtain more confident results.
Owing to time and world limitation, we have restricted our protocol to the study of beta-glucans on microglial cells. However, other cells from the immune system such as macrophages or T cells can be easily easily during this protocol. For the first time, these protocols combine two different area of knowledge.
First, the use of biotechnological procedure to purify specific products, and second, the use of cell culture techniques to test their immunomodulatory properties.
