Picogreen dna quantification kit

Following method published in Chuammitri et al. (2009 (link)), isolated bovine PMNs were plated at 1 × 10⁶ in quadruplicate in 96 well plates for NET stimulation and quantitation. Cells were allowed to settle in plates for 1 h at 37°C, 5% CO₂, at which time bacteria MOI 10 or stimulant was added to quadruplicate wells and incubated at 37°C for 2 h. Replicate wells were set up in the same manner, with the addition of 100 U/ml DNase I (RQ1 RNase free-DNase, Promega). After incubation, micrococcal nuclease (Worthington Biochemical Corporation) was added to a final concentration of 0.1 U/ml and incubated for an additional 30 min at 37°C. 5 mM EDTA was added to stop the reaction. Plates were centrifuged at 400 × g for 2.5 min at 25°C, and 100 μl of supernatant removed. DNA concentration of the supernatant was quantified (PicoGreen DNA quantification kit, Molecular Probes) and read in a fluorescence plate reader (SpectraMAX GeminiXS, Molecular Devices, excitation 492 nm, emission 520 nm; with SOFTMax PRO software). Data is expressed as mean of replicate wells ng/ml DNA in supernatant. Data was analyzed from three independent experiments using cells isolated from six animals. Data was analyzed using Graphpad Prism 6 Software and one-way ANOVA with Tukey's multiple comparisons post-test.

LAP with a diameter of 50 nm and a thickness of 7 nm was purchased from Zhejiang Institute of Geologic and Mineral Resources Co., Ltd. (Hangzhou, China). Eagle's Minimal Essential Medium (α-MEM), Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), phosphate buffer saline (PBS), penicillin, and streptomycin were purchased from Gibco (Carlsbad, CA). β-Glycerophosphate (β-GP), ascorbic acid, resazurin, p-nitrophenyl phosphate, and p-nitrophenol standard were from Sigma (St. Louis, MO). Reporter Lysis Buffer and Picogreen DNA quantification kit were from Molecular Probes, Inc. (Eugene, OR). rMSC and heparin-stabilized pig blood was kindly provided by Shanghai First People's Hospital (Shanghai, China). All other chemicals were from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) and used as received. Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with resistivity higher than 18 MΩ·cm.

Participants were instructed to use the investigator-provided stool collection kit to collect a stool sample at home. The sample was placed in a sterile conical tube, placed in a biohazard bag, and stored in the participant’s freezer until the date and time of the pre-determined pick-up from their home (not more than 3 days from time of collection). Samples were picked up by research personnel and immediately transported on ice to the University of Calgary Faculty of Kinesiology and stored at −80 °C degrees until analysis. Gut microbial profiling was conducted as previously described [47 (link),48 (link)]. Briefly, bacterial DNA was extracted from ~60 mg of fecal matter according to the manufacturer’s instructions, using the FastDNA Spin Kits for feces (MP Biomedicals, Lachine, QC, Canada). Bacterial DNA was then diluted to 4 ng/uL using the PicoGreen DNA quantification kit (Invitrogen, Carlsbad, CA, USA) and stored at −20 °C until sequencing. Microbial composition was assessed using the MiSeq Illumina platform, which amplified the V3 and V4 regions of the 16S rRNA gene (Illumina, San Diego, CA, USA). Extracted bacterial DNA samples were sequenced at the University of Calgary’s Centre for Health Genomics and Informatics (Calgary, AB, Canada).

Microbial community DNA was extracted from P. placomus following the protocol described in Sunagawa, Woodley & Medina (2010) (link). Rather than grinding the sample, the protocol was modified by clipping a small piece (one polyp and its attached piece of central skeleton) from each octocoral sample using sterile forceps and shears and then placing it into a bead tube supplied with the PowerPlant DNA extraction kit (MO BIO, Carlsbad, CA, USA). Polyps were always collected from the part of the coral sample furthest from the end grasped during collection to limit any contamination from the ROV claw. The Sunagawa protocol was further modified by increasing the proteinase K (Ambion, Grand Island, NY, USA) incubation to 90 min from the protocol’s stated 60 min. Extracted DNA was quantified using the PicoGreen DNA quantification kit (Invitrogen, Grand Island, NY, USA) and the presence of bacterial DNA was confirmed by PCR amplification of 16S rRNA genes, using primers Eco8F (Edwards et al., 1989 (link)) and 1492R (Stackebrandt & Liesack, 1993 ), AmpliTaq Gold polymerase, and thermal cycled as follows: 1 cycle of 95 °C for 15 min; 30 cycles of 95 °C for 1 min, 54 °C for 1 min, and 72 °C for 2 min; and a final extension of 72 °C for 10 min.

Fecal matter was collected prior to, and at the end of, the 4 week treatment period, in order to longitudinally measure the microbiota profile of each mouse prior to and following THC or vehicle treatment. Fecal samples were stored at -80°C until analysis. Microbial DNA was extracted from samples using FastDNA Spin Kit for Feces (MP Biomedicals, Lachine, QC) and quantified using PicoGreen DNA quantification kit (Invitrogen, Carlsbad, CA). Microbiota was quantified using qPCR as previously described [26 (link)]. Mice fed HFD excreted fecal pellets that were observed to be drier, smaller and fewer in number than those from the lean mice. As such it was not possible to collect enough fecal matter for microbiota analysis from all the DIO mice without removing them from their home cage for a prolonged period of time. This stressor may have had detrimental effects on their feeding behavior. As a result, the sample size for some groups is low.