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7 protocols using pyrex

1

Cytotoxicity Assay in 96-well Microplates

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Instruments used were an evaporator Rotavapor RV 10 Digital V connected to heating bath HB digital and RV 10.1 set of glassware vertical (IKA Id. no. 0010004799), chemical glassware (Iwaki Pyrex), autoclave (all American type 75X), Biological Safety Cabinet (BSC) type-2, microplate reader (Infinite M200 Pro, Tecan), microplate reader filter 450–490 nm, inverted microscope (Zeiss), sterilized 96-well microplates, multichannel pipettes (8 or 12 channels: 10–100 μL), CO2 incubator (Heracell VIOS 250i, Thermo Scientific), and hemocytometer (cell counter).
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2

Physicochemical Characterization of Fresh Cow Milk

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Fresh cow milk was organoleptically tested to determine the taste, smell and color. The pH value of fresh cow milk was determined using a digital pH meter. The pH meter was firstly calibrated at pH 4 and 10 with buffer standards solution. Milk as much as 50 mL was then placed in a beaker, the calibrated pH meter was then inserted and the obtained pH value was recorded. Viscosity was measured using a Brookfield viscometer (Brookfield DVII, USA) with spindle no.4 and 3 rpm rotation speed, results recorded in centipoise (cP) after 60 seconds of shearing. The density of milk was measured using pycnometer 25 mL (IWAKI pyrex). Protein and fat contents of the milk were also investigated for further characterization.
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3

Culturing and Growth Kinetics of S. pyogenes

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S. pyogenes M1T1 strain 5448 (accession no. CP008776) was isolated from a patient with toxic shock syndrome and necrotizing fasciitis and considered to be a genetically representative globally disseminated clone associated with the invasive infections (65 (link)). S. pyogenes strain 5448 was cultured in a screw-cap glass tube (Pyrex; Iwaki Glass, Tokyo, Japan) filled with Todd-Hewitt broth (BD Biosciences, San Jose, CA) supplemented with 0.2% yeast extract (THY) (BD Biosciences) at 37°C in an ambient atmosphere. For growth measurements, overnight cultures of S. pyogenes strain 5448 were back diluted 1:50 into fresh THY and grown at 37°C, with growth monitored by measuring optical density at 600 nm (OD600).
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4

Tyrosinase Inhibition by C. latifolia Extracts

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The inhibitory activity against tyrosinase of root, stem, and leaf extracts of C. latifolia was determined colorimetrically based on the modification of previous methods [30 (link), 31 (link)]. The reaction mixture in a 96-well microplate (Iwaki Pyrex) consisted of 80 µL of phosphate buffer (50 mM, pH 6.5), 40 µL of extract solution (1–500 µg/mL), 40 µL of L-DOPA solution (4 mM), and 40 µL of mushroom tyrosinase solution (75 U/mL). The solution mixture was shaken for 60 seconds and incubated for 30 minutes at 25°C. The absorbance was measured using a microplate reader (Promega GloMax) at 490 nm. The blank solution in phosphate buffer was prepared in the same way as the sample solution. Control samples and blanks were made without the addition of the tyrosinase enzyme. The following equation was used to calculate the percentage of tyrosinase inhibition: inhibition%=ABCDAB×100%, where A is the absorbance of the blank solution with enzymes (blank), B is the absorbance of the blank solution without enzymes (blank control), C is the absorbance of the sample solution with enzymes (sample), and D is the absorbance of the sample solution without enzymes (control sample). The IC50 value was calculated using a linear regression equation generated from plots of sample concentration versus percentage of inhibition (% inhibition).
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5

Isolation and Quantification of Streptococcus pyogenes RNA

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Bacterial cultures during exponential phase (OD600 = 0.5-0.6), early stationary phase (OD600 = 1.2), or decline phase (overnight culture) were centrifuged and immediately placed into RNAprotect Bacteria Reagent (Qiagen) prior to RNA isolation. In the RNA isolation from S. pyogenes cultured within CDM, bacterial cultures during exponential phase (OD600 = 0.5-0.6) were centrifuged, resuspended into CDM, incubated in a screw cap glass tube (Pyrex; Iwaki Glass, Tokyo, Japan) for 1 h at 37°C, and and immediately placed into RNAprotect Bacteria Reagent. S. pyogenes was resuspended into lysing Matrix B microtubes containing 0.1-mm silica spheres (Qbiogene, Carlsbad, CA, USA) with RLT lysis buffer (RNeasy Mini Kit; Qiagen), and homogenized at 6,500 rpm for 60 s using the MagNA Lyser (Roche Molecular Diagnostic, Mannheim, Germany). RNA was isolated from the lysate with RNeasy Mini Kit according to the manufacturer’s guidelines, and then cDNA was synthesized using a Superscript VILO cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time reverse transcription PCR analysis was performed using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) and Toyobo SYBR green RT-PCR master mix kit (Toyobo Life Science, Osaka, Japan). Data for 16S rRNA or rpoB were used as the internal control. Primers used for qPCR are listed in Table S2.
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6

Streptococcus pyogenes Culturing and Manipulation

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Streptococcus pyogenes M1T1 strain 5448 (accession: CP008776.1) was isolated from a patient with toxic shock syndrome and necrotizing fasciitis that is genetically representative of a globally disseminated clone associated with invasive S. pyogenes infections (Kansal et al., 2000 (link)). S. pyogenes strains were grown at 37°C in a screw-cap glass tube (Pyrex; Iwaki Glass, Tokyo, Japan) filled with Todd-Hewitt broth (BD Biosciences, San Jose, CA, USA) supplemented with 0.2% yeast extract (BD Bioscience) (THY broth) in an ambient atmosphere and standing cultures. To obtain cultures for experiments and observe pH change, overnight cultures of S.pyogenes were back diluted 1:50 into fresh THY broth or phenol red broth (Sigma Aldrich, St Louis, MO, USA) supplemented with 30 mM arginine. CFUs were determined by plating diluted samples on THY blood agar.
Escherichia coli strain XL-10 Gold (Agilent Technologies, Santa Clara, CA, USA) was used as a host for derivatives of plasmids pSET4s (Takamatsu et al., 2001 (link)) and pQE30 (Qiagen, Hilden, Germany). E. coli strains were cultured in Luria-Bertani medium (Nacalai Tesque, Kyoto, Japan) at 37°C with agitation. For selection and maintenance of strains, antibiotics were added to the medium at the following concentrations: spectinomycin, 100 μg/mL for S. pyogenes and E. coli: carbenicillin, 100 μg/mL for E. coli.
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7

RNA-seq Analysis of S. pyogenes Transcriptome

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Bacterial cultures during exponential phase were centrifuged, resuspended into CDM, and incubated in a screw-cap glass tube (Pyrex; Iwaki Glass, Tokyo, Japan) for 1 h at 37°C. RNA samples of S. pyogenes were obtained after incubation, as described above. RNA integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). For RNA-seq, bacterial RNA was treated for rRNA removal using a Ribo-Zero rRNA removal kit (Illumina Inc., San Diego, CA, USA). RNA-seq libraries were created using a TruSeq RNA Sample Prep kit, v2 (Illumina Inc.), according to the manufacturer’s recommendations. Libraries were sequenced using Illumina HiSeq 2500 systems, with 75-bp single-end reads. RNA-seq reads were mapped against the S. pyogenes strain 5448 genome using the commercially available CLC Genomics workbench, v. 9.5.2 (CLC Bio, Aarhus, Denmark). Global analyses of RNA-seq expression data were performed using iDEP (Ge et al., 2018 (link)), with the FPKM value of each sample. We classified the differentially expressed genes (DEGs) into functional categories based on the bacterial bioinformatics database and analysis resource PATRIC (Wattam et al., 2017 (link)). The heatmap was visualized by use of the web tool ClustVis (Metsalu and Vilo, 2015 (link)) with default parameters.
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