The H1 hESC line was acquired from the WiCell Research Institute (Madison, WI), propagated on Matrigel-coated vessels and cultured in mTeSR (both from StemCell Tech). The H7 hESC cell line (WiCell Research Institute) was propagated and maintained on Matrigel coated vessels in X-Vivo10 Medium (Lonza), supplemented with 80ng/ml FGF2 and 0.5ng/ml TGFBI (R&D Systems). hESC were cultured in feeder-free, serum-free conditions and cells were passaged at approximately 80% confluence by treatment with 5mg/ml Collagenase IV for 5 min, followed by wash with PBS and reconstitution in an appropriate volume of medium. If an estimation of an accurate cell number of starting material was required prior to the differentiation process, after collagen treatment one representative cell culture vessel was sacrificed and hESC cells disaggregated with 0.25% Trypsin-EDTA (all from Life Technologies). 10% FBS (PAA) in RPMI 1640 medium (Life Technologies) was then used to stop trypsinization and the number of total and viable cells determined using a NucleoCounter YC-100 (Chemometec). The cells were then washed with PBS, scraped in medium and passaged into new Matrigel coated vessels at a cell density of 0.6x105cells/cm2. hESC culture media were changed daily.
Nucleocounter yc 100
Manufactured by ChemoMetec
Sourced in Denmark
The NucleoCounter YC-100 is a compact and automated cell counter designed for quick and accurate cell counting. It utilizes fluorescence microscopy technology to determine the total cell count and viability of a sample. The device provides fast and reliable results, making it a useful tool for various cell-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type xi, by Merck Group (2 mentions)
P2 bio gel column, by Bio-Rad (2 mentions)
X vivo 10 medium, by Lonza (1 mentions)
Tgfbi, by R&D Systems (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (1 mentions)
H1 hesc line, by WiCell (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Axiovert 40 cfl, by Zeiss (1 mentions)
L 15 medium, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Biocoll, by Harvard Bioscience (1 mentions)
Cobe 2991 cell processor, by Terumo BCT (1 mentions)
Ham s f10 based medium, by Lonza (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Diff quik, by Thermo Fisher Scientific (1 mentions)
Series 200 lc instrument, by PerkinElmer (1 mentions)
13 protocols using nucleocounter yc 100
1
Feeder-Free Culture of Human Embryonic Stem Cells
2
Islet Cell Purification and Characterization
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Islet cell preparations were characterized by their number of beta cells after purification and after a 1- to 5-day culture period [determined after a minimum of two washing cycles to eliminate damaged cells and cell debris, by combining cell number (nuclear count assay in duplicate samples; NucleoCounter YC-100; ChemoMetec, Allerød, Denmark) and percentage of insulin positive cells (immunocytochemistry; >103 cells counted)] [7 (link), 22 (link)]. Yield immediately post purification was also expressed as islet equivalent (IEQ), calculated using a volume based method after dithizone (DTZ) staining [23 (link), 24 ]. Only preparations with these data available after purification were included for analysis. Cellular insulin content was determined and expressed as μg insulin / 106 beta cells [22 (link)].
3
Transfection and Imaging of Chinook Salmon Cells
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Chinook salmon embryonic cells (CHSE-214) were seeded in standard flasks (Nunc™) with L-15 medium (Invitrogen) containing penicillin (60 μg ml−1), streptomycin (100 μg ml−1), 1% non-essential amino acids (NEAA, Gibco) and 8% fetal bovine serum (FBS). The CHSE-214 cells were incubated at 20 °C for 1 week. Cells were then washed twice with 10 mL PBS before adding 1.5 mL trypsin (Sigma). Loosened cells were re-suspended in L-15 medium (8% FBS, 1% NEAA, without antibiotics) and counted using a Nucleocounter YC-100 (Chemometec, Allerod, Denmark). Transfections of plasmids were performed using the Invitrogen NeonTM Transfection System according to the supplier’s protocols (Invitrogen). The cells were either transfected with pTagRFP-T-bet, pTagRFP-GATA-3, pTagRFP-TGF-β or pTagRFP-N, expressing plasmids in separate wells (1 × 105 cells per well). Mock-transfected cells were used as the controls. After 12 h of incubation in the transfection medium, the cells were gently washed and received L-15. After 48 h, the cells were fixed with 4% formaldehyde (w/v) (Thermo Scientific, Waltham, MA, USA) for 30 min, then DAPI (4′,6-diamidino-2-phenylindole ) (Sigma) was used for nucleic acid staining, according to the manufacturer’s protocols. Micrographs were obtained with an inverted fluorescence microscope equipped with DAPI-365 and Texas Red 530-585 filters (Zeiss Axiovert 40 CFL).
4
Culturing Ethanol-Tolerant Saccharomyces cerevisiae
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Saccharomyces cerevisiae strain (NCYC 2826) was obtained from the National Collection of Yeast Cultures (NCYC, Norwich, UK) and exhibits high ethanol tolerance of 15–20% (v/v). The strain was sub-cultured from a slope culture by inoculation into 1 L of Difco™ Yeast and Mould (YM) broth (Fisher Scientific UK Ltd., Loughborough, UK) containing 0.3% (w/v) yeast extract, 0.3% (w/v) malt extract, 0.5% (w/v) peptone and 1% (w/v) dextrose (Elliston et al., 2013 (link)). Yeast was grown in this medium for 2 days at 25 °C and subsequently stored at 4 °C for up to 1 month before use (Elliston et al., 2013 (link)). Before yeast inoculation of the fermentation mix, the cultured yeast medium was centrifuged (3000 rpm, 5 min, Centrifuge 5810 R, Eppendorf UK Ltd., Stevenage, UK), the supernatant (YM media) discarded, and the yeast cells resuspended in nitrogen base (ForMedium™, Formedium Ltd, Hunstanton, UK). The total viable yeast cells were measured by using a cell count reader (NucleoCounter® YC-100™, ChemoMetec, Allerød, Denmark). The standard yeast culture contained 8.0 × 107 cells mL−1 of S. cerevisiae NCYC2826.
5
Islet Cell Isolation and Characterization
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Islet cells were isolated using a modification of the automated Ricordi method17
and purified by continuous gradient with Biocoll (Biochrom, Berlin, Germany) and a cooled COBE 2991 cell processor (Terumo BCT, Lakewood, CO, USA). After isolation and purification, the cell preparations were cultured in cell culture flasks T175 (Sarstedt, Nümbrecht, Germany) at 37°C in a humidified incubator (5% CO2) in a Ham’s F10 based medium (Lonza, Bazel, Switzerland).
Preparations were characterized immediately after purification and after a 1- to 5-day culture period by their beta cell number, insulin content, and insulin purity, as described previously17
. Beta cell number was calculated from the total nuclear count (NucleoCounter YC-100; ChemoMetec, Allerod, Denmark) and the percentage of insulin positive cells [immunocytochemistry with guinea pig anti-insulin (1/2,000, in-house produced) on 1.5 µm araldite sections; >2 × 103 cells counted; pictures were captured using Nikon Eclipse Ti microscope and analyzed with NIS-Elements AR v5.21 software (Nikon Europe, Amsterdam, The Netherlands;Fig. 1 )]. Yield immediately post purification was also expressed as islet equivalent (IEQ), calculated using a volume based method after dithizone staining19
.
and purified by continuous gradient with Biocoll (Biochrom, Berlin, Germany) and a cooled COBE 2991 cell processor (Terumo BCT, Lakewood, CO, USA). After isolation and purification, the cell preparations were cultured in cell culture flasks T175 (Sarstedt, Nümbrecht, Germany) at 37°C in a humidified incubator (5% CO2) in a Ham’s F10 based medium (Lonza, Bazel, Switzerland).
Preparations were characterized immediately after purification and after a 1- to 5-day culture period by their beta cell number, insulin content, and insulin purity, as described previously17
. Beta cell number was calculated from the total nuclear count (NucleoCounter YC-100; ChemoMetec, Allerod, Denmark) and the percentage of insulin positive cells [immunocytochemistry with guinea pig anti-insulin (1/2,000, in-house produced) on 1.5 µm araldite sections; >2 × 103 cells counted; pictures were captured using Nikon Eclipse Ti microscope and analyzed with NIS-Elements AR v5.21 software (Nikon Europe, Amsterdam, The Netherlands;
.
6
BALF Cell Differential Analysis
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BALF was centrifuged at 200×g for 5 minutes, and supernatant was removed for cytokine measurement. Pelleted cells were resuspended and counted by NucleoCounter YC-100 (ChemoMetec, Allerød, Denmark) automated cell number counting. 100 µl of cell suspension was then loaded onto a glass slide using a disposable sample funnel and cytocentrifuged at 10×g for 3 minutes in a Shandon Cytospin 2 centrifuge. Slides were air dried for 20 minutes, fixed in methanol for 20 minutes, stained with Diff Quik (Fisher Scientific, Loughborough, UK), and mounted in DPX Mountant (Fluka BioChemika/Sigma Aldrich, UK). Differential counts for neutrophils and monocytes were then performed by light microscopy at 20× magnification using an EVOS FL microscope (Peqlab, Sarisbury Green, UK).
7
Optimized Ethanol Production via Consolidated Bioprocessing
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A sample of each pretreated substrate was suspended in 10 mL solution with a final concentration of 5 % substrate in nitrogen base (Formedium, Hunstanton, UK) and held in 20mL screw-topped glass vials. Both cellulases (36 FPU cellulase/g substrate) and a concentrated yeast inoculum were added to each vial and incubated for 96 h, 40 °C.
The yeast inoculum used was a robust thermo-tolerant yeast (Saccharomyces cerevisiae, strain NCYC 2826, National Collection of Yeast Cultures, Norwich, UK) grown from a slope culture, inoculating 1 L of yeast mould (YM) broth (3 d, 25 °C) before centrifuging, discarding the supernatant and partially reconstituting the yeast in nitrogen base. The final solutions contained 3.83 × 107 viable cells/mL when inoculated—assayed using a NucleoCounter® YC-100™ (ChemoMetec, Denmark). SSFs were conducted as three independent replicates, and the ethanol released from a cellulase + yeast control was subtracted from each sample.
Ethanol concentrations were quantified using HPLC using a Series 200 LC instrument (PerkinElmer, UK) equipped with an Aminex HPX-87P carbohydrate analysis column (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). The mobile phase used was ultrapure water (0.6 mL/min) and concentration quantified using a refractive index (RI) detector, comparing absorbance to a set of ethanol standards.
The yeast inoculum used was a robust thermo-tolerant yeast (Saccharomyces cerevisiae, strain NCYC 2826, National Collection of Yeast Cultures, Norwich, UK) grown from a slope culture, inoculating 1 L of yeast mould (YM) broth (3 d, 25 °C) before centrifuging, discarding the supernatant and partially reconstituting the yeast in nitrogen base. The final solutions contained 3.83 × 107 viable cells/mL when inoculated—assayed using a NucleoCounter® YC-100™ (ChemoMetec, Denmark). SSFs were conducted as three independent replicates, and the ethanol released from a cellulase + yeast control was subtracted from each sample.
Ethanol concentrations were quantified using HPLC using a Series 200 LC instrument (PerkinElmer, UK) equipped with an Aminex HPX-87P carbohydrate analysis column (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). The mobile phase used was ultrapure water (0.6 mL/min) and concentration quantified using a refractive index (RI) detector, comparing absorbance to a set of ethanol standards.
8
Yeast Chromosomal DNA Isolation and PFGE
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Yeast strains were propagated in YPM at 20 °C to an OD600 >1 and then harvested by centrifugation (3,000×g, 5 min, 4 °C). Supernatants were decanted, and cells were resuspended in 10 ml of 4 °C 50 mM EDTA (pH 8). Cell concentrations were determined with a Nucleocounter® YC-100™ (ChemoMetec) and 1.2 × 108 cells were placed in each sample plug. Sample plugs were prepared with the CHEF Genomic DNA Plug Kit for Yeast (Bio-Rad) according to the manufacturer’s instructions.
Sample plugs were loaded into the wells of a 1.0 % pulse field certified agarose (Bio-Rad) gel. PFGE was performed at 14 °C in 0.5× TBE buffer [89 mMTris, 89 mM boric acid, 2 mM EDTA (pH 8)]. A CHEF Mapper XA pulsed field electrophoresis system (Bio-Rad) was used with the following settings: 6 V/cm in a 120° angle, pulse length increasing linearly from 26 to 228 s, and total running time of 38 h. A commercial chromosome marker preparation from S. cerevisiae strain YNN295 (Bio-Rad) was used for molecular mass calibration. After electrophoresis, the gels were stained with ethidium bromide and scanned with Gel Doc XR+ imaging system (Bio-Rad).
Sample plugs were loaded into the wells of a 1.0 % pulse field certified agarose (Bio-Rad) gel. PFGE was performed at 14 °C in 0.5× TBE buffer [89 mMTris, 89 mM boric acid, 2 mM EDTA (pH 8)]. A CHEF Mapper XA pulsed field electrophoresis system (Bio-Rad) was used with the following settings: 6 V/cm in a 120° angle, pulse length increasing linearly from 26 to 228 s, and total running time of 38 h. A commercial chromosome marker preparation from S. cerevisiae strain YNN295 (Bio-Rad) was used for molecular mass calibration. After electrophoresis, the gels were stained with ethidium bromide and scanned with Gel Doc XR+ imaging system (Bio-Rad).
9
Isolation and Characterization of Implanted Cell Tissue
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At PT‐week 20, implanted devices were retrieved and opened to isolate tissue content and disperse it to an aggregate suspension (<500 μm diameter) using type XI collagenase (1 mg/ml, Sigma–Aldrich). After washing, samples were taken for analysis of cell number (NucleoCounter YC‐100, ChemoMetec, Denmark), composition and functions as previously described 4 , 5 . Cell composition following dispersion was determined by manual count in immunocytochemistry on cells fixed in glutaraldehyde and embedded in araldite. Sections were stained using the same antibodies as described above. Insulin secretory responsiveness to glucose was determined in a perifusion assay. A sample with 500–700 × 103 cells was loaded on a P2‐Bio‐gel column (Bio‐Rad, CA) and perifused for 60‐minutes at 2.5 mmol/l glucose (basal), before pulsing with 5, 10, or 20 mmol/l glucose, with or without 10 nmol/l glucagon 4 . Perifusate was collected in 1 minute fractions and assayed for insulin 4 . Data were expressed per 103 β cells as determined in the sample that was added to the column.
10
Insulin Secretion Dynamics in Tissue Aggregates
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Retrieved devices were opened and their tissue content removed for collagenase
dispersion into aggregates (<500 µm diameter) (type XI collagenase, 1 mg/ml,
Sigma-Aldrich, USA). After washing, samples were taken for determining cell
number (NucleoCounter YC-100, ChemoMetec, Denmark) and composition7
before loading 0.5 to 1 x 106 cells on a P2-Bio-gel column
(Bio-Rad, USA) for assessing insulin secretory responses during
perifusion5 ,7 . One-minute fractions were collected and assayed for
insulin; data were expressed per 103 beta cells as determined in the
sample before loading.
dispersion into aggregates (<500 µm diameter) (type XI collagenase, 1 mg/ml,
Sigma-Aldrich, USA). After washing, samples were taken for determining cell
number (NucleoCounter YC-100, ChemoMetec, Denmark) and composition7
before loading 0.5 to 1 x 106 cells on a P2-Bio-gel column
(Bio-Rad, USA) for assessing insulin secretory responses during
perifusion5 ,7 . One-minute fractions were collected and assayed for
insulin; data were expressed per 103 beta cells as determined in the
sample before loading.
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