The plasma protein binding assay was conducted with the Rapid Equilibrium Dialysis (RED) (Wang and Williams, 2013 (link)). 3 μM FB23-2 was added to rat plasma, which were vortexed well before placing 100 μl in the red chamber of the RED device (Thermo, USA). 300 μl of PBS (pH 7.4) was added to the corresponding white chamber, the base plate was covered with a gas-permeable membrane, and then incubated in a CO2 incubator (MCO-18AC, Panasonic, Japan) set at 250 rpm and 37 °C for 4 hr. Samples collected from both chambers were analyzed with LC-MS/MS to determine the compound concentrations (C0 - initial concentration, Cf - ultrafiltrate and Cp - plasma). Percentage of protein binding (PPB) using the RED device was calculated with the formula, PPB (%) = (1- Cf /Cp) × 100%. The experiments were performed in triplicate.
Mco 18ac
Manufactured by Panasonic
Sourced in Japan, United Kingdom
The MCO-18AC is a laboratory equipment product manufactured by Panasonic. It is designed to provide a controlled environment for various laboratory applications. The core function of this product is to maintain precise temperature and humidity levels within an enclosed space.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Defibrinated horse blood, by Thermo Fisher Scientific (2 mentions)
Yeast extract, by Liofilchem (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Microflex lt sh, by Bruker (2 mentions)
Red device, by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (1 mentions)
Puromycin, by Selleck Chemicals (1 mentions)
Streptomycin, by Boster Bio (1 mentions)
Metformin, by Selleck Chemicals (1 mentions)
Penicillin, by Boster Bio (1 mentions)
Hitransg p, by Genechem (1 mentions)
Starch, by ITW Reagents (1 mentions)
Glycerol, by ITW Reagents (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Mem a, by Capricorn (1 mentions)
Hos 1a, by Capricorn (1 mentions)
Mc3t3 e1 pre osteoblasts, by American Type Culture Collection (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
17 protocols using mco 18ac
1
Rapid Equilibrium Dialysis Assay for Protein Binding
2
HUVEC and 4T1 Cell Culturing
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HUVEC and mouse breast cancer cell line 4T1 (4T1) were purchased from the American Type Culture Collection. DMEM was supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. Cells were cultured and maintained in the above-mentioned growth medium and incubated overnight at 37°C in 5% CO2 at 95% humidity (MCO-18AC, Panasonic).
3
Endometrial Cancer Cell Culture and YAP1 Knockout
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Human type I endometrial carcinoma Ishikawa and RL95-2 cells were previously donated by the Cancer Biology Research Center of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Cells were cultured in Dulbecco's modified Eagle medium (DMEM)-high glucose (HyClone, United States) containing 10% (v/v) fetal bovine serum (FBS, Gibco, United States), 100 U/mL penicillin (Boster, China), and 100 μg/mL streptomycin (Boster, China) at 37°C in a humidified atmosphere with 5% CO2 in an incubator (MCO-18AC, Panasonic, Japan). Metformin (S1950, Selleck, China) was dissolved in ddH2O and then diluted to a suitable concentration. YAP1-knockout (YAP1-KO) lentivirus (LV-YAP1-KO) based on the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was purchased from Genechem, China. Ishikawa cells were transfected with LV-YAP-KO using HitransG P (Genechem, China). Following selection with 5 μg/ml puromycin (CL13900, Selleck, China) for 72 h, single-cell clones were sorted, expanded, and validated as YAP1-KO Ishikawa cells by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis.
4
Culturing Gardnerella Bacterial Species
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G. vaginalis UM137, G. piotii UM035, G. leopoldii UGent 09.48 and G. swidsinskii GS 9838-1 were grown on Columbia Agar Base medium (Liofilchem, Roseto degli Abruzz, Italy) supplemented with 5% (v/v) defibrinated horse blood (Oxoid Ltd., Hampshire, UK) for 48 h [7 (link)]. For each experiment, the bacterial species were grown in Brain Heart Infusion (BHI, Liofilchem) supplemented (sBHI) with 2% (w/v) gelatine (Liofilchem), 0.1% (w/v) starch (Panreac, Barcelona, Spain) and 0.5% (w/v) yeast extract (Liofilchem) and incubated for 24 h at 37 °C and 10% CO2 (Panasonic MCO-18AC, Bracknell, UK).
5
Gardnerella Strains Culturing and Maintenance
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Various strains from the genus Gardnerella were analyzed in this study. Gardnerella sp. UM241, previously recovered from patients diagnosed with BV93 (link), G. vaginalis UM137, G. piotii UM035, G. leopoldii UGent 09.48, and G. swidsinskii GS 9838-1, previously identified by MALDI-TOF15 (link),94 (link), were used herein. The strains were kept frozen in Brain Heart Infusion medium (BHI, Liofilchem, Roseto degli Abruzz, Italy) with 23% (v/v) of glycerol (Panreac, Barcelona, Spain) and plated in Columbia Blood Agar (CBA) [Columbia Base Agar medium (Liofilchem) supplemented with 5% (v/v) of defibrinated horse blood (Oxoid Ltd, Hampshire, UK)] and incubated for 48 h at 37 °C and 10% CO2. For each experiment, planktonic cells were grown in BHI supplemented (sBHI) with 2% (w/v) gelatin (Liofilchem), 0.1% (w/v) starch (Panreac) and 0.5% (w/v) yeast extract (Liofilchem) and incubated for 24 h at 37 °C and 10% CO2 (CO2 Incubator MCO-18AC, Panasonic, Bracknell, UK).
6
Cell Viability Assay of Fibroblasts
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The cell viability of normal mice fibroblasts (CCL-1TM, NCTC clone 929, American Type Culture Collection—ATCC, Manassas, VA, USA) was evaluated according to ISO 10993-5, Annex C [86 ]. The cells were cultured at 37 °C under sterile conditions and humidified atmosphere in a CO2 incubator (Panasonic MCO-18AC, Kadoma, Osaka, Japan) supplied continuously with 5% CO2. Cells were maintained in the culture medium MEM (#MEM-A, Capricorn®, Munich, Germany) with addition of 10% heat inactivated horse serum (#HOS-1A, Capricorn®, Munich, Germany) and 2 mM of L-glutamine (#G7513, Sigma-Aldrich, Steinheim, Germany). Cells were seeded in 96-well plates in concentration 1 × 104/100 µL medium/well as recommended in ISO 10993-5, Annex C [86 ]. The tested concentrations of the extracts varied from 0.02 up to 1 mg/mL in two-fold serial dilutions. The incubation period was 72 h. The cell viability was measured on a microplate reader ELx800 (BioTek Instruments, Inc., Winooski, VT, USA) at λ = 540 nm/ref690 nm.
7
Osteoblast Differentiation of MC3T3-E1 Cells
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MC3T3-E1 pre-osteoblasts were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in α-minimum essential medium without L-ascorbic acid (L-AA) (WELGEME, Inc., Gyeonggido, Korea), containing 10% fetal bovine serum and 1× antibiotic–antimycotic (Thermo Fisher Scientific, Waltham, MA, USA), in a CO2 incubator MCO-18AC (Panasonic, Osaka, Japan). Osteoblast differentiation was initiated by using an osteogenic supplement medium OS containing 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA) and 50 μg/mL L-AA (Sigma-Aldrich) in a CO2 incubator. The OS was changed every 2 days for differentiation.
8
Cultivation of Mouse Fibroblast Cell Line
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The cell line CCL-1™ (mouse fibroblasts, NCTC clone 929, ATCC—American Type Culture Collection, Manassas, Virginia, USA) recommended in Annex C of ISO 10993-5 (ISO 10993-5:2009 2017) for evaluation of in vitro cytotoxicity was cultured in sterile cell culture flasks and controlled environment (incubator Panasonic MCO-18AC, Japan) at 37 °C, 5% CO2 and approx. 95% humidity. The cultivation medium MEM was supplemented with 2 mM l -glutamine, 10% heat-inactivated horse serum, 105 Units/l penicillin G sodium and 100 mg/l streptomycin sulphate. Cells were sub-cultivated at a seeding density of 1 × 104 cells/cm2 1–3 times per week after reaching 80–90% confluence. Sequentially, applied solutions of 0.05% EDTA in PBS (1–2 ml, 5–10 min) and 0.25% (w/v)/0.53 mM trypsin/EDTA (1–2 ml, 5–10 min) were used for the detachment of the cell monolayer and cell separation.
9
Evaluating EPS-LP2 Cytotoxicity in Macrophages
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Following Zhou’s approach (15 (link)), RAW264.7 cells were cultivated in Dulbecco’s modified eagle medium (DMEM, including 10% fetal bovine serum and 1% streptomycin-penicillin) and incubated at 37°C in an incubator (MCO-18 AC, Panasonic, Osaka, Japan) with 5% CO2. MTT assay was applied to identify the cytotoxic effect of EPS-LP2 on macrophages (16 (link)). Cells were firstly treated with EPS-LP2 solution at different concentrations (0, 50, 100, 200, and 400 μg/ml, 100 μL) for 2 h. Cells were then stimulated with DMEM containing 1 μmol/L H2O2 as a blank and treated with vitamin C (Vit C) (100 μg/ml) as a positive control. MTT (10 μL) was included in each cell and incubated for 2 h at 37°C in an incubator with 5% CO2 incubator. The sample absorbance values were measured at 570 nm with a microplate reader (SMR60047, USCNK).
10
Cell Culture in RPMI-1640 with Supplements
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SCCVII and C26 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic (#09366-44, Nacalai Tesque, Kyoto, Japan) in 5% CO2 in an incubator (MCO-18AC, Panasonic Healthcare) at 37 °C.
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