To screen human isolates for isoalloLCA production, isolates were retrieved from stock plates from our human fecal screen library (Paik et al., 2021 (link)) and cultured in 600 μL Cullen-Haiser Gut (CHG) media (Hall et al., 2017 (link)), which consists of BHI supplemented with 1 % BBL vitamin K1-hemin solution (BD Biosciences, 212354), 1% trace minerals solution (ATCC, MD-TMS), 1 % trace vitamins solution (ATCC, MD-VS), 5% fetal heat-inactivated bovine serum (FBS) (Genesee Scientific, 25–514), 1 g/L cellobiose (Sigma-Aldrich, C7252), 1 g/L maltose (Sigma-Aldrich, M5895) and 1 g/L fructose (Sigma-Aldrich, F0127), containing 0.5% (w/v) arginine (Sigma-Aldrich, A5006) for 48 hours at 37°C in 96-well plates. Each isolate, as well as the negative controls, was then diluted 1:10 in new media containing 100 μM LCA (Sigma-Aldrich) or 100 μM 3-oxoLCA (Steraloids) for an additional 48 hours. 0.2 mL cultures were harvested and extracted for bile acid analyses (see below). This experiment was conducted once per substrate for all isolates from the original eleven library plates. Following bile acid analysis, we performed 16S rRNA sequencing on isoalloLCA-producing strains; subsequently, their function was verified in culture tubes in triplicate.
C7252
Manufactured by Merck Group
The C7252 is a versatile lab equipment product from the Merck Group. It is designed to perform core laboratory functions, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information is not available.
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2 protocols using c7252
1
Screening Human Gut Isolates for isoalloLCA Production
2
Isolation and Characterization of Piriformospora indica Cell Wall Extract
Check if the same lab product or an alternative is used in the 5 most similar protocols
Piriformospora indica cell-wall extract (PiCWE) was prepared as described by Vadassery et al., 2009 (link). In brief, the mycelia from 14-d-old liquid cultures were homogenized, filtered using nylon membranes, and washed three times with water, twice with chloroform/methanol (1:1), and finally twice with acetone. The mycelial cell wall material obtained was dried at room temperature, suspended in water, and autoclaved for 30 min at 121 °C. It was then filter-sterilized using a 0.22-μM filter, and 50 µl of the resulting extract was used per seedling root for experiments. The active elicitor of PiCWE was recently identified as cellotriose (CT; Johnson et al., 2018 (link)). CT (Sigma, C1167) and cellobiose (Sigma, C7252) were used for some experiments.
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Piriformospora indica cell-wall extract (PiCWE) was prepared as described by Vadassery et al., 2009 (link). In brief, the mycelia from 14-d-old liquid cultures were homogenized, filtered using nylon membranes, and washed three times with water, twice with chloroform/methanol (1:1), and finally twice with acetone. The mycelial cell wall material obtained was dried at room temperature, suspended in water, and autoclaved for 30 min at 121 °C. It was then filter-sterilized using a 0.22-μM filter, and 50 µl of the resulting extract was used per seedling root for experiments. The active elicitor of PiCWE was recently identified as cellotriose (CT; Johnson et al., 2018 (link)). CT (Sigma, C1167) and cellobiose (Sigma, C7252) were used for some experiments.
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