Depending on the number of available faecal pellets, samples were re-suspended in either 450 or 900 μl of sterile phosphate buffer saline (Sigma-Aldrich, UK) and used to produce tenfold serial dilutions (neat - 10-4). The samples were then vortexed for 30 s and mixed using on a shaker at 1600 rpm. An aliquot of each dilution (100 μl) was plated onto Brain Heart Infusion (BHI) (Oxoid, UK) agar supplemented with mupirocin (50 mg/l) (Sigma-Aldrich, UK), l -cysteine hydrochloride monohydrate (50 mg/l) (Sigma-Aldrich, UK) and sodium iodoacetate (7.5 mg/l) (Sigma-Aldrich, UK) and incubated in an anaerobic cabinet for 48–72 h (Baker Ruskinn, UK). Three colonies from each dilution were randomly selected and streaked to purity on BHI agar supplemented with l -cysteine hydrochloride monohydrate (50 mg/l). Pure cultures were stored in cryogenic tubes at −80 °C.
Sterile phosphate buffer saline
Manufactured by Merck Group
Sourced in United States
Sterile phosphate buffer saline is a balanced salt solution used for various laboratory applications. It maintains the physiological pH and osmolarity of biological samples. The solution is sterilized to prevent contamination.
Automatically generated - may contain errors
Lab products found in correlation
Sodium iodoacetate, by Merck Group (1 mentions)
Mupirocin, by Merck Group (1 mentions)
L cysteine hydrochloride monohydrate, by Merck Group (1 mentions)
Rnalater solution, by Merck Group (1 mentions)
Sterile container, by Sarstedt (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
5 protocols using sterile phosphate buffer saline
1
Isolation and Cryopreservation of Anaerobic Bacteria
2
Culturable Microbial Diversity in Restoration Soils
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The culturable microbial diversity of both field soils from the restoration site and the healthy native reference site at Mount Annan and the autoclaved soil mix was investigated via culturing on tryptic soy agar plates (Edwards Group Holdings, Narellan, Australia). Three samples of each soil type were serially diluted in sterile phosphate buffer-saline (Sigma-Aldrich, Missouri, United States) to concentrations of 1:1000, 1:2000, 1:4000, 1:8000, 1:16,000 and 1:32,000 (m/v). All dilutions and a buffer only control were plated in triplicate and incubated at 25 °C for five days. Only the 1:16,000 and 1:32,000 dilutions were plated for the Mount Annan field soil samples while for the autoclaved soil mix all dilutions were plated to account for the anticipated diminished microbial diversity and richness after autoclaving. Morphotypes were categorized according to their visual characteristics (form, elevation, margin, surface, opacity, and pigmentation).
3
Intestinal Tissue and Digesta Sampling
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All birds from the control and the best performing laminarin group, 300 ppm inclusion level, were euthanized on d 35 by cervical dislocation and one bird per pen was randomly selected for sample collection. Euthanasia was completed by a trained individual in a separate room away from sight and sound of the other birds. For gene expression analysis, duodenal, jejunal and ileal tissue samples were rinsed in sterile phosphate buffer saline (Sigma-Aldrich, St. Louis, MO), cut into 1 cm2 tissue sections using a sterile scalpel. These were stored overnight at room temperature in RNAlater solution (Sigma-Aldrich, St. Louis, MO), and subsequently at −20°C prior to RNA extraction. Digesta were collected using gentle digital pressure from both caeca per bird and placed into sterile containers (Sarstedt, Nümbrecht, Germany). This was then snap frozen on dry ice and stored at −80°C for subsequent microbial analysis (QPCR and high-throughput sequencing).
4
Isolation of Mesenchymal Stem Cells from Human Amniotic Fluid
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Back-up flasks of human amniotic fluid cell (hAFCs) samples (n = 5) were obtained during weeks 16–22 of gestation from the Human Genetics Laboratory of Anatomy Department, Faculty of Medicine, Chiang Mai University. After being analyzed, normal karyotype cells were then used in this study. This study was approved by the Research Ethics Committee of the Faculty of Medicine, Chiang Mai University on March 13th, 2018 (no. ANA-2561-05343).
To isolate MSCs, the direct adherent method was performed as it has been previously explained [28 (link)]. The hAFCs were washed with sterile phosphate buffer saline (PBS) (Sigma-Aldrich, USA) and trypsinized with 0.25% trypsin-EDTA (Gibco, USA). After observed, detached cells were then diluted with basal medium consisting of Dulbecco's Modified Eagle Medium (DMEM) (Gibco, USA) – high glucose with 10% fetal bovine serum (FBS) (Gibco, South America), gentamycin (T.P. Drug Laboratories, Thailand) and Pen Strep (penicillin and streptomycin) (Gibco, USA). The hAFCs were plated in non-pyrogenic polyprolene 25 cm2 (Corning®, NY, USA) at 37 °C and 5 % CO2 at 95% humidity. Upon reaching 80% confluence, the cells were sub-cultured to remove non-adherent cells. After observing the presence of a fibroblast-like morphology, the cells were then used in the experiments.
To isolate MSCs, the direct adherent method was performed as it has been previously explained [28 (link)]. The hAFCs were washed with sterile phosphate buffer saline (PBS) (Sigma-Aldrich, USA) and trypsinized with 0.25% trypsin-EDTA (Gibco, USA). After observed, detached cells were then diluted with basal medium consisting of Dulbecco's Modified Eagle Medium (DMEM) (Gibco, USA) – high glucose with 10% fetal bovine serum (FBS) (Gibco, South America), gentamycin (T.P. Drug Laboratories, Thailand) and Pen Strep (penicillin and streptomycin) (Gibco, USA). The hAFCs were plated in non-pyrogenic polyprolene 25 cm2 (Corning®, NY, USA) at 37 °C and 5 % CO2 at 95% humidity. Upon reaching 80% confluence, the cells were sub-cultured to remove non-adherent cells. After observing the presence of a fibroblast-like morphology, the cells were then used in the experiments.
5
Surface Friction Testing of Tantalum
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The details of the SFT set-up and the sequences of the SFT processing of pure (99.95 wt%) Ta were given in ref. 19 . The given processing parameters for this study were as follows: 500 N in load, 0.2 m/s in sliding velocity, 100 μm in offset displacement perpendicular to the sliding direction after each sliding direction, and 100 in cycle. Square samples of 10 × 10 mm2 cut from both the SFT treated and untreated Ta sheets were ultrasonically cleaned in acetone, ethanol, and distilled water for 10 min each and dried in air. For in vitro biocompatibility studies, all samples were sterilized with alcohol for 3 h and rinsed twice with sterile phosphate buffer saline (PBS; Sigma, USA) before cell seeding.
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