Immunobeads (Irvine Scientific, California, CA, USA), bovine blood (Japan Bio Serum Co. Ltd., Fukuyama, Japan), and heparin-treated human blood (Rockland Immunochemicals Inc., Pennsylvania, PA, USA) were purchased. Bovine blood cells and human blood cells were stained using the Wright–Giemsa stain kit (ScyTec Laboratories Inc., Utah, UT, USA) and Diff-Quik stain kit (Sysmex, Kobe, Japan), respectively. The running buffer was prepared using 0.5% (w/v) bovine serum albumin (BSA; Nacalai Tesque, Kyoto, Japan), and 2 mM EDTA (Dojindo, Kumamoto, Japan) in 10 mM PBS (Wako, Tokyo, Japan).
Ethylene diamine tetraacetic acid (edta)
Manufactured by Dojindo Laboratories
Sourced in Japan, United States
EDTA is a chelating agent used in various laboratory applications. It functions by forming stable complexes with metal ions, including calcium and magnesium. EDTA is commonly used in analytical techniques, buffer solutions, and sample preparation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Fujifilm (4 mentions)
Sodium octane sulfonate, by Dojindo Laboratories (3 mentions)
Sodium deoxycholate, by Fujifilm (2 mentions)
Ethylene glycol tetraacetic acid (egta), by Dojindo Laboratories (2 mentions)
Dm irb, by Leica (2 mentions)
Trypsin, by Merck Group (2 mentions)
Collagenase, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Triton x 100, by Nacalai Tesque (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Nacalai Tesque (1 mentions)
Phosphate buffered saline (pbs), by Fujifilm (1 mentions)
Diff quik stain kit, by Sysmex (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Triton x 100, by MP Biomedicals (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Middlebrook 7h9 medium, by BD (1 mentions)
Protein assay rapid kit, by Fujifilm (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
26 protocols using ethylene diamine tetraacetic acid (edta)
1
Immunostaining of Blood Cells
2
Preparation of Decellularized Amniotic Membrane
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Human fetal membranes were harvested from patients undergoing cesarean delivery at the Tokyo Medical and Dental University Hospital. Written informed consent was obtained from all subjects. This research protocol was approved by the clinical research ethics committee at the Tokyo Medical and Dental University (TMDU). The amniotic membrane was washed, cut into small pieces, and incubated for 1 h at 37 °C in 0.02% ethylenediamine tetraacetic acid (EDTA, Dojindo, Tokyo, Japan) in phosphate-buffered saline (PBS). After incubation, the chorion was mechanically removed from the amnion using a cell scraper and tweezers. The resulting amnion was stored in DMEM containing 50% glycerol (WAKO) at −80 °C until use. Cell components of the amnion were removed by high hydrostatic pressure treatment as previously reported11 12 (link).
3
Dissection and Homogenization of Brain Regions
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Seven coronal sections of the brain at 2 mm thickness were made, and the third section from the rostral side was collected for the following dissection. The coronal section was dissected into two regions, cerebral cortex and striatum (S), for both hemispheres. The cerebral cortex was further divided into the non-ischemic (N) and ischemic (C) regions. Samples of the six regions in the coronal section were defined as: LN, region of the left cerebral cortex corresponding with the non-ischemic region in the right hemisphere; LC, region of the left cerebral cortex corresponding with the ischemic region in the right hemisphere; LS, striatum in left hemisphere; RN, non-ischemic region of the cerebral cortex in right hemisphere; RC, ischemic region of the cerebral cortex in right hemisphere; and RS, striatum in right hemisphere. Dissected sections were homogenized in lysis buffer (1% Triton X-100; MP Biomedicals (Santa Ana, CA, USA), 0.1% sodium deoxycholate; Wako (Osaka, Japan), 1% EDTA; DOJINDO (Kumamoto, Japan), Complete protease inhibitor cocktail; Roche (Basel, Switzerland), PhosStop phosphatase inhibitor cocktail; Roche, in 50 mM Tris-buffered saline) with Ultrasonic Liquid Processor Q 125 (QSONICA) for 20 s at 4 °C. The homogenate was centrifuged at 15,000 × g for 5 min at 4 °C, and the supernatant was collected for immunoblotting.
4
Characterization of Bacterial Antimicrobial Mechanisms
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The following agents were used: ATP (Sigma Aldrich Co., St. Louis, MO; MP Biomedicals, Solon, OH, USA, Calbiochem Co., La Jolla, CA, and Roche Diagnostic Co., Indianapolis, IN), ADP (Sigma), AMP (Sigma), adenosine (Sigma), benzoylbenzoyl ATP (Sigma), oxidized ATP (Sigma), suramin (Wako, Tokyo, Japan), MIA (methyl isobutyl amiloride, Wako), DIDS (4,4′- Diisothiocyanatostilbene-2,2′-disulfonic acid, Sigma), EDTA (Dojindo, Tokyo, Japan), EGTA (Dojindo), dipyridyl (Sigma), CAS (Chrome Azurol S, Sigma), E. coli S17-1 and pK18mobSacB (kindly provided by Dr. F. Taguchi, Okayama University), Instagene matrix (Bio-Rad, Hercules, CA), KOD-Plus (Toyobo, Osaka, Japan), Wizard SV Gel and PCR cleanup system (Promeg, Madison, WI), Ligation High (Toyobo), Protein Assay Rapid Kit (Wako), [14C] isoleucine (Moravek Biochemicals, Inc, Brea, CA), [14C]uracil (Moravek Biochemicals, Inc.), α-defensin-1 (Peptide institute, Inc, Osaka, Japan.), cathepsin G (Sigma), vancomycin (Wako), clarithromycin (Taisho-Toyama Pharmaceutical Co., Tokyo), rifampin (Daiichi Sankyo Co., Tokyo), and ethambutol (Sigma), gatifloxacin (Wako), FLUOS (Sigma), LB medium (Invitrogen, San Diego, CA), M9 medium (prepared by our laboratory), Heart infusion agar (Eiken Chemical Co., Tokyo, Japan), Middlebrook 7H9 medium (Becton Dickinson, Cockeysville, MD), and Middlebrook 7H11 medium (Becton Dickinson).
5
Platelet Preparation and Activation Assay
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Platelet preparation and activation assays were performed according to the methods of Goodall and Appleby45 (link) with minor modifications. Mouse blood was collected under deep anaesthesia in a tube containing 20 unit/ml heparin (Mitsubishi Tanabe Pharma, Osaka, Japan). The whole blood was centrifuged for 5 min at 500 × g to prepare the platelet rich plasma (PRP). The PRP was centrifuged again for 8 min at 300 × g. After the addition of 0.5 μM prostacyclin (Cayman Chemical, Ann Arbor, MI, US), the PRP was centrifuged for 5 min at 1300 × g. The platelet preparation was incubated for 30 min at 37 °C with PBS containing 0.1% bovine serum albumin (Roche Diagnostics, Basel, Switzerland); 2 mM EDTA (Dojindo, Kumamoto, Japan); 0.02 unit/ml apyrase (New England BioLabs, Hertfordshire, UK); and 0.5 μM prostacyclin after washing with the same buffer. The prepared platelet containing fraction was analysed by flow cytometry using Aria II (Becton Dickinson) after staining with anti-CD41 FITC (eBioscience, San Diego, CA, US) and anti-CD62P APC (eBioscience). Platelet activation was evaluated by the reactivity of anti-CD62P APC against the platelet fraction, which was gated with forward/side scatter characteristics and anti-CD41 FITC positive fractions.
6
Chondrocyte Culture Analysis Protocol
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Cell lysates of cultured chondrocytes were collected from 12-well polystyrene culture
plates every 2 weeks during the culture period for quantification of total DNA and GAGs
content. After culture media was removed and washed with PBS, papain solution containing
300 µg/ml papain (Sigma-Aldrich, St. Louis, MO, USA)
in 20 mM Na2HPO4 (Wako), 2 mM dithiothreitol (Wako) and 1 mM EDTA
(Dojindo) was added to perform cell digestion. Digested chondrocytes were incubated with
papain solution for 18 hr at 60°C. The total DNA content was quantified by Hoechst 33258
assay (Wako) with a calf thymus DNA as a standard (Sigma-Aldrich) and detected with
350/460 nm filter set. Total GAGs content quantification was performed with cell lysate
and culture media to evaluate both GAGs accumulation and GAGs released in culture media.
Dimethylmethylene blue (DMMB) assay (Sigma-Aldrich) was used with chondroitin sulphate as
a standard (Wako) and absorbance was measured at 525 nm, then normalized with DNA content.
Microplate reader (Infinite M200 Pro, Tecan, Männedorf, Switzerland) was used for
measurement in both assays.
plates every 2 weeks during the culture period for quantification of total DNA and GAGs
content. After culture media was removed and washed with PBS, papain solution containing
300 µg/ml papain (Sigma-Aldrich, St. Louis, MO, USA)
in 20 mM Na2HPO4 (Wako), 2 mM dithiothreitol (Wako) and 1 mM EDTA
(Dojindo) was added to perform cell digestion. Digested chondrocytes were incubated with
papain solution for 18 hr at 60°C. The total DNA content was quantified by Hoechst 33258
assay (Wako) with a calf thymus DNA as a standard (Sigma-Aldrich) and detected with
350/460 nm filter set. Total GAGs content quantification was performed with cell lysate
and culture media to evaluate both GAGs accumulation and GAGs released in culture media.
Dimethylmethylene blue (DMMB) assay (Sigma-Aldrich) was used with chondroitin sulphate as
a standard (Wako) and absorbance was measured at 525 nm, then normalized with DNA content.
Microplate reader (Infinite M200 Pro, Tecan, Männedorf, Switzerland) was used for
measurement in both assays.
7
Fecal DNA Extraction Protocol
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Fecal DNA was extracted based on a previous report with some modifications [57 (link)]. Approximately 0.2 g of fecal sample was suspended in a 50-mL Falcon tube containing 20 mL of PBS. The suspension was washed with 10 mL of PBS and debris was removed with a 100-µm mesh nylon filter (Corning Inc., New York, NY, USA). After centrifuging the filter at 4000 rpm for 20 min at 4 °C, each precipitate was suspended in 1.5 mL of TE10 buffer composed of 10 mM Tris-HCl (FUJIFILM Wako Pure Chemicals Co., Ltd.) and 10 mM EDTA (Dojindo, Tokyo, Japan). After the suspension was transferred to another microtube and centrifuged at 10,000 rpm for 5 min at 4 °C, each precipitate was resuspended in 0.8 mL of TE10 buffer. DNA was extracted using 1 mL of PCI (Invitrogen, Carlsbad, CA, USA), and 0.1 mL of lysozyme (FUJIFILM Wako Pure Chemicals Co., Ltd.) and 0.2 mL of achromopeptidase (FUJIFILM Wako Pure Chemicals Co., Ltd.) was used to isolate the DNA. The DNA was treated with RNase (Promega Corp., Madison, WI, USA) and then purified by precipitation with 20% polyethylene glycol (PEG) solution (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). The DNA was rinsed with 70% ethanol and dissolved in 50 μL of TE buffer.
8
Isolation of Peripheral Blood Mononuclear Cells
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Peripheral blood samples (10 mL) were obtained from 21 healthy adult volunteers and 25 lung cancer patients after approval of the institutional review board of Nagasaki University Hospital and with written informed consent. All methods were performed in accordance with the guidelines and regulations of Nagasaki University Hospital. Peripheral blood samples were heparinized and diluted with Dulbecco's phosphate buffered saline (-) (PBS, Nissui Pharmaceutical Co., Ltd. Taito-ku, Tokyo, Japan). The diluted blood samples were loaded on Ficoll-PaqueTM PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and peripheral blood mononuclear cells (PBMCs) were purified by gradient centrifugation. The cells were washed two times with PBS, then resuspended in 80 μL of PBS containing 0.5% BSA fraction V (Nacalai Tesque Inc., Nakagyo-ku, Kyoto, Japan) and 2 mM EDTA (Dojindo Laboratories, Kamimashiki-gun, Kumamoto, Japan) and the cell suspension was placed on ice.
9
Citric Acid-Based Mobile Phase Preparation
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Citric acid monohydrate (8.84g; MW210.14), and 3.10g of sodium acetate (MW82.03) in 800 ml of MilliQ Ultrapure fresh water (>18.2M2/cm) and 200 ml of HPLC grade methanol were added. EDTA (MW372.24; 0.005g) and sodium octane sulfonate (0.220 g), both from Dojindo Laboratories, Rockville, MD, were added.
10
SYBR Green Assay for Plasmodium falciparum
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SYBR green assays were performed by modifying the methods from previous studies46 (link),63 (link),64 (link) for evaluating P. falciparum growth. Briefly, P. falciparum was cultured for ≈96 h (two cycles), the P. falciparum-RBC burst was collected in sterilized water, and the erythrocyte membranes were removed by centrifugation (18,000 × g, 15 min). Aliquots (100 μL) of the supernatant from each sample were mixed with 100 μL of the assay reagents; SYBR green (Molecular Probes Inc., Eugene, OR, USA), Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany), saponin (Sigma), 20 mM-Tris (Invitrogen) and 500 mM EDTA (Dojindo, Kamimashiki-gun, Japan) for 1 h at room temperature. The fluorescence intensity of the SYBR G was measured by Molecular Devices Spectra Max M5 (Molecular Devices, LLC., San Jose, CA, USA) (EX = 521 nm, EM = 591 nm).
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