Based on cytotoxicity results for the 2 h pulse treatment with ricin toxin, cells were seeded at 5 × 105 cells/ml in collagen‐coated 96‐well plates and were treated with ricin at a dose approximately equal to the EC90 value on the ricin dose curves. Approximately 18 h after seeding, LSECs and KCs were treated with 50 ng/ml of ricin, and J774E cells were treated with 100 ng/ml of ricin for 2 h. Concurrently, cells were treated with either 1 mg/ml of yeast α‐mannan (Sigma–Aldrich), 0.1 M lactose (Thermo Fisher), or a mixture of yeast α‐mannan and lactose. Cell viability was assessed 24 h later using CellTiter‐GLO reagent and a Spectramax L Microplate reader. Cell viability was normalized to cells treated with medium only and background noise was reduced by subtracting the average of wells treated only with ricin at a dose equivalent to the EC90 value.
Lactose
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United Kingdom, United States
Lactose is a disaccharide sugar composed of galactose and glucose. It is commonly found in milk and dairy products. Lactose serves as a source of energy and nutrients for various applications in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
M17 broth, by Thermo Fisher Scientific (4 mentions)
Yeast α mannan, by Merck Group (2 mentions)
Yeast α mannan, by Thermo Fisher Scientific (2 mentions)
Sucrose, by Thermo Fisher Scientific (2 mentions)
Sorbitol, by Thermo Fisher Scientific (2 mentions)
Fructose, by Thermo Fisher Scientific (2 mentions)
Maltose, by Thermo Fisher Scientific (2 mentions)
Sharpe broth, by Merck Group (1 mentions)
Lm17 broth, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Forskolin, by STEMCELL (1 mentions)
Sodium butyrate, by Merck Group (1 mentions)
Forskolin, by Santa Cruz Biotechnology (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Rnazol rt, by Molecular Research Center (1 mentions)
Hypoxia chamber, by STEMCELL (1 mentions)
Barium hydroxide, by Thermo Fisher Scientific (1 mentions)
Zinc sulfate, by Thermo Fisher Scientific (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
25 protocols using lactose
1
Evaluating Ricin Toxin Cytotoxicity
2
Ricin Cytotoxicity Mitigation Assay
3
Culturing Streptococcus thermophilus
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Pure bacterial cultures of S. thermophilus 285 were obtained from Victoria University culture collection (Werribee, VIC, Australia). Stock cultures were stored in cryobeads at −80°C. Prior to each experiment the cultures were propagated in M17 broth (Oxoid, Denmark) with 20 g/L lactose and incubated at 37°C under aerobic conditions. Bacteria were also cultured in M17 agar (1.5% w/v agar) with 20 g/L lactose (Oxoid, Denmark), to assess characteristics, morphology, purity and gram-positive confirmation [1 ].
4
Culturing S. thermophilus 285 for Research
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Pure bacterial cultures of S. thermophilus 285 (ST285) were obtained from the Victoria University culture collection (Werribee, Vic, Australia). Stock cultures were stored in cryobeads at −80 °C. Prior to each experiment, the cultures were propagated in M17 broth (Oxoid, Denmark) with 20 g/L lactose and incubated at 37 °C under aerobic conditions. Bacteria were also cultured on M17 agar (1.5% w/v agar) with 20 g/L lactose (Oxoid, Denmark) to assess the characteristics, morphology, purity, and Gram-positive confirmation [1 (link)].
5
Culturing and Characterizing ST285 Bacteria
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Pure bacterial cultures of ST285 were obtained from Victoria University culture collection (Werribee, VIC, Australia). Stock cultures were stored in cryobeads at -80° C. Prior to each experiment the cultures were propagated in M17 broth (Oxoid, Denmark) with 20 g/L lactose and incubated at 37° C under aerobic conditions. In order to confirm gram-positivity and assess purity, morphology and characteristics, bacteria were cultured in M17 agar (1.5 % w/v agar) with 20 g/L lactose (Oxoid, Denmark) as well [1] .
6
Isolation and Characterization of Ur Starter Culture
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Single-colony isolates of the complex starter culture Ur used in this study were collected from LM17 agar plates (Oxoid, Basingstoke UK; 1.5% wt/vol) supplemented with lactose (Oxoid; 0.5% wt/vol; Terzaghi and Sandine, 1975) and Reddy agar plates as described previously by Erkus et al. (2013) . Individual isolates were characterized and classified into 8 genetic lineages by AFLP typing (Kütahya et al., 2011) : Lactococcus lactis ssp. cremoris (lineages 1, 3, 5, 6, and 7), Lactococcus lactis ssp. lactis biovar diacetylactis (lineages 2 and 4), and Leuconostoc mesenteroides ssp. cremoris (lineage 8). Similarity of the genetic profiles of <90% was considered as the cut-off for defining separate genetic lineages. Isolates were maintained as glycerol stocks at -80°C and reactivated, in the case of lactococci, in LM17 broth (Oxoid) with addition of 0.5% (wt/vol) lactose (Oxoid) and, for Leuconostoc strains, in de Man, Rogosa, and Sharpe broth (Merck, Schiphol-Rij, the Netherlands).
7
Chicken Macrophage and Jejunal Explant Responses to Butyrate, Forskolin, and Lactose
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Chicken HD11 macrophage cells [19 (link),23 (link)] were cultured in complete RPMI 1640 (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Lonza, Walkersville, MD, USA) at 1 × 106 cells/well in 12-well culture plates overnight, followed by treatment in triplicate with 2 mM sodium butyrate [19 (link),23 (link)] (MilliporeSigma, St. Louis, MO, USA), 5 μM forskolin [23 (link)] (Santa Cruz Biotechnology, Dallas, TX, USA), or 0.1 M lactose (Thermo Fisher Scientific, Nazareth, PA, USA) separately or in combination. After incubation at 37 °C and 5% CO2 for 24 h, cells were lysed with RNAzol RT (Molecular Research Center, Cincinnati, OH, USA) for total RNA extraction according to the manufacturer’s protocol.
Chicken jejunal explants were prepared and cultured as previously described [23 (link),31 (link)]. Briefly, the jejunal segments were collected from one- to two-week-old chickens, cultured in 6-well plates, treated in triplicate with 4 mM sodium butyrate [23 (link)], 5 μM forskolin [23 (link)], and 0.1 M lactose alone or in combination, and incubated in Hypoxia Chamber (StemCell Technologies, Vancouver, BC, Canada) filled with 95% O2 and 5% CO2 at 37 °C for 24 h, followed by centrifugation and RNA extraction.
Chicken jejunal explants were prepared and cultured as previously described [23 (link),31 (link)]. Briefly, the jejunal segments were collected from one- to two-week-old chickens, cultured in 6-well plates, treated in triplicate with 4 mM sodium butyrate [23 (link)], 5 μM forskolin [23 (link)], and 0.1 M lactose alone or in combination, and incubated in Hypoxia Chamber (StemCell Technologies, Vancouver, BC, Canada) filled with 95% O2 and 5% CO2 at 37 °C for 24 h, followed by centrifugation and RNA extraction.
8
Measuring Intestinal Lactase and Sucrase
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Intestinal lactase and sucrase activities were measured as previously described (33 (link), 34 (link)). Briefly, mucosal homogenates were prepared and incubated with 0.6 M lactose (Thermo Fisher Scientific) or 0.3 M sucrose (Thermo Fisher Scientific) buffer (dissolved in 0.0625 M maleate buffer) for 60 min at 37°C. The reaction was stopped by the addition of 2.0% zinc sulfate (Thermo Fisher Scientific) and 1.8% barium hydroxide (Thermo Fisher Scientific), and the amount of glucose released was detected via a glucose oxidase reagent (Thermo Scientific). Enzyme activity was in μmoles glucose per minute per gram of protein. Protein was measured in intestinal homogenates via the Bradford method (Bio-Rad).
9
Lactose Fermentation Assay for Bacterial Isolates
10
Microbial Growth Media and Nitrogen Sources
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Culture media (Brain heart infusion (BHI), M17, MRS, tryptic soy broth (TSB) and LB media) and inorganic nitrogen sources ((NH4)2SO4, NH6PO4, NH4NO3 and NH4Cl) were purchased from Merck (Darmstadt, Germany). Carbon sources (fructose, glucose, galactose, sucrose, lactose, maltose, sorbitol and mannitol) were obtained from Fisher Chemical (Loughborough, United Kingdom), while organic nitrogen sources (yeast extract, meat extract, peptone and soytone) were from BD (Franklin Lakes, NJ, USA).
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