At passage 6, cells were treated with Accutase to dissociate, and 250,000 cells were added to each 15-mL conical polypropylene tube. Cells were centrifuged at 1500 RPM for 5 minutes and resuspended in 500 μL chondrogenic medium (CM) containing DMEM-HG, 50 μg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich), 40 μg/mL DL-proline (Sigma-Aldrich), 1% ITS+ Premix (Becton-Dickinson, Franklin Lakes, NJ), 1% P/S and with either no growth factors, or with 100 ng ml−1 BMP2 (Peprotech) and/or 10 ng ml−1 TGF-β3 (Peprotech). Cells were then pelleted at 500xg for 5 minutes. All pellets were incubated at 37°C in 5% CO2 for 21 days, with CM refreshed every 2–3 days.
Dl proline
Manufactured by Merck Group
Sourced in United States, Germany
DL-proline is an amino acid that can be used as a laboratory reagent. It is a racemic mixture of the D- and L-enantiomers of the amino acid proline.
Automatically generated - may contain errors
Lab products found in correlation
Dl alanine, by Merck Group (3 mentions)
Ethanol, by Merck Group (2 mentions)
4 methyl 1 pentanol, by Merck Group (2 mentions)
Oasis max cartridge 6 cc with stationary phase, by Waters Corporation (2 mentions)
Tgf β3, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid 2 phosphate, by Merck Group (1 mentions)
Its premix, by BD (1 mentions)
Bmp 2, by Thermo Fisher Scientific (1 mentions)
5977a mass selective detector, by Agilent Technologies (1 mentions)
Hp innowax fused silica capillary column, by Agilent Technologies (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
N n dimethylformamide (dmf), by Thermo Fisher Scientific (1 mentions)
Ethyl acetate, by Avantor (1 mentions)
Benzoic acid, by Thermo Fisher Scientific (1 mentions)
1 methylimidazole, by Merck Group (1 mentions)
1 chlorobutane, by Thermo Fisher Scientific (1 mentions)
1 methyl 2 pyrrolidone, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Merck Group (1 mentions)
Pivalic acid, by Merck Group (1 mentions)
Dl phenylalanine, by Merck Group (1 mentions)
6 protocols using dl proline
1
Chondrogenic Differentiation of Cells
2
Preparation of Amino Acid Standards
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Ethanol (>99.9%) was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) for standards preparation. 4-Methyl-1-pentanol, an internal standard (ISTD) compound, was also obtained from Sigma-Aldrich. dl -alanine and dl -proline were purchased from Sigma-Aldrich and Oasis MAX cartridge 6 cc with stationary phase was purchased from Waters (Waters, Milford, MA, USA).
3
GC-MS Analysis of Amino Acids
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GC-MS analysis was performed using a HP 7890B gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with a 5977A mass selective detector (MSD) (Agilent Technologies) and a multi-purpose sampler MPS 2 (Gerstel, Mülheim an der Ruhr, Germany). HP-innowax fused silica capillary column (30 m length × 0.25 mm internal diameter × 0.25 μm film thickness; Agilent Technologies) was used with helium, a carrier gas, at a constant flow rate of 1 mL/min. Injection volume was 1.0 μL with split mode (20:1). The oven temperature program started at 40 °C initially and was held for 3 min, then was raised to 200 °C at 5 °C/min and held at 200 °C for 3 min. Post run was held at 180 °C for 2 min. Inlet, detector transfer line and mass source temperatures were 250, 280, 230 °C, respectively. MS was operated in the electron impact (EI) ion source mode at 70 eV with a scan range of 25~350 a.m.u. Ethanol (>99.9%) was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) for standard preparation. 4-Methyl-1-pentanol, an internal standard (ISTD) compound, was also obtained from Sigma-Aldrich. dl -alanine and dl -proline were purchased from Sigma-Aldrich and Oasis MAX cartridge 6 cc with stationary phase was purchased from Waters (Waters, Milford, MA, USA).
4
Synthesis and Characterization of Polysulfone Membranes
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Polysulfone (PSU, Mw 35,000 Da in transparent pellet form, Sigma Aldrich, St. Louis, MO, USA), N,N-Dimethylformamide (DMF, 99% Fisher, Loughborough, UK), and 1-Methyl-2-pyrrolidone (NMP, 99% Acros Organics, New Jersey, NY, USA) were used for membrane preparation.
For the synthesis of 1-butyl-3-methylimidazolium chloride, 1-chlorobutane (99% Acros Organics, Geel, Belgium), 1-methylimidazole (99% Sigma-Aldrich, St. Louis, MO, USA), acetonitrile (CAN, 99.8% Fisher, Loughborough, UK), and ethyl acetate (99.9% VWR, Fontenay-Sous-Bois, France) were used. The task-specific ILs were prepared by the ion exchange process using Amberlite IRA-402(Cl) ion exchange resin (Alfa-Aesar, Kandel, Germany), Sodium hydroxide (NaOH, 97% Fisher, Geel, Belgium), and acidic compounds: benzoic acid (99% Acros Organics, New Jersey, USA), pivalic acid (99% Sigma-Aldrich, Steinheim, Germany), DL-proline (99% Sigma-Aldrich, St. Louis, USA), formic acid (99% Merck, Darmstadt, Germany), and malonic acid (99% Alfa-Aesar, Kandel, Germany)
The membrane gas solubility was evaluated with CO2 (99.995%) and N2 (99.999%) gases purchased from Linde (Barcelona, Spain).
For the synthesis of 1-butyl-3-methylimidazolium chloride, 1-chlorobutane (99% Acros Organics, Geel, Belgium), 1-methylimidazole (99% Sigma-Aldrich, St. Louis, MO, USA), acetonitrile (CAN, 99.8% Fisher, Loughborough, UK), and ethyl acetate (99.9% VWR, Fontenay-Sous-Bois, France) were used. The task-specific ILs were prepared by the ion exchange process using Amberlite IRA-402(Cl) ion exchange resin (Alfa-Aesar, Kandel, Germany), Sodium hydroxide (NaOH, 97% Fisher, Geel, Belgium), and acidic compounds: benzoic acid (99% Acros Organics, New Jersey, USA), pivalic acid (99% Sigma-Aldrich, Steinheim, Germany), DL-proline (99% Sigma-Aldrich, St. Louis, USA), formic acid (99% Merck, Darmstadt, Germany), and malonic acid (99% Alfa-Aesar, Kandel, Germany)
The membrane gas solubility was evaluated with CO2 (99.995%) and N2 (99.999%) gases purchased from Linde (Barcelona, Spain).
5
Capillary Electrophoresis Separation of Amino Acids
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All reagents were of analytical grade. FMOC-Cl, β-CD, pentane, sodium tetraborate, sodium hydroxide, glycine, d -glutamic acid, d -histidine, d -threonine, l -alanine, l -arginine, l -asparagine, l -aspartic acid, l -cysteine, l -glutamic acid, l -glutamine, l -histidine, l -isoleucine, l -leucine, l -lysine, l -methionine, l -proline, l -serine, l -threonine, l -tryptophan, l -tyrosine and l -valine, dl -alanine, dl -arginine, dl -asparagine, dl -aspartic acid, dl -cysteine, dl -glutamic acid, dl -histidine, dl -isoleucine, dl -leucine, dl -lysine, dl -phenylalanine, dl -proline, dl -serine, dl -tryptophan, and dl -valine were from Sigma-Aldrich (Steinheim, Germany). Isopropanol, dl -methionine, dl -tyrosine, sodium dodecyl sulfate, and acetonitrile were supplied by Fluka (Steinheim, Germany). Water was deionized and purified with a Milli-Q purification system (Millipore, Belford, NJ, USA).
The optimal BGE was 40 mM sodium tetraborate (adjusted to pH 9.5 with 1 M sodium hydroxide) containing 15% (v/v) isopropanol, 30 mM sodium dodecyl sulfate (SDS), and 30 mM β-CD. The BGE was filtered prior to use through 0.45-μm pore size disposable nylon filters from VWR (Amsterdam, The Netherlands). Stock solutions (3 mM) of AAs were prepared in 0.2 M sodium tetraborate (pH 9.5).
The optimal BGE was 40 mM sodium tetraborate (adjusted to pH 9.5 with 1 M sodium hydroxide) containing 15% (v/v) isopropanol, 30 mM sodium dodecyl sulfate (SDS), and 30 mM β-CD. The BGE was filtered prior to use through 0.45-μm pore size disposable nylon filters from VWR (Amsterdam, The Netherlands). Stock solutions (3 mM) of AAs were prepared in 0.2 M sodium tetraborate (pH 9.5).
6
Enzyme Stabilization in Lyophilized Powder
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Lyophilized powder of peroxidase
from HRP, (type I, 89.63 U/mg solid, CAS 9003-99-0) was purchased
from Sigma-Aldrich (St. Louis, Missouri, USA) and used without further
purification. D-(+)-xylose (≥99%, CAS 58-86-6), glycerol (≥99.5%
CAS 56-81-5), D-sorbitol (≥98%, CAS 50-70-4), DL-proline (99%,
CAS 609-36-9), phenol-4-sulfonic acid sodium salt dihydrate (PSA,
98%, CAS 10580-19-5), 4-aminoantipyrine (4-AAP, ≥ 99%, CAS
83-07-8) and hydrogen peroxide 30% solution (CAS 7722-84-1) were purchased
from Sigma-Aldrich (St. Louis, Missouri, USA). Trehalose dihydrate
(CAS 6138-23-4) was kindly provided by Hayashibara Co., LDA (Okayama,
Japan). Betaine anhydrous (>97%, CAS 107-43-7) was obtained from
TCI
(Tokyo, Japan) and sucrose (CAS 57-50-1) was purchased from Cmd Chemicals
(Funchal, Portugal).
from HRP, (type I, 89.63 U/mg solid, CAS 9003-99-0) was purchased
from Sigma-Aldrich (St. Louis, Missouri, USA) and used without further
purification. D-(+)-xylose (≥99%, CAS 58-86-6), glycerol (≥99.5%
CAS 56-81-5), D-sorbitol (≥98%, CAS 50-70-4), DL-proline (99%,
CAS 609-36-9), phenol-4-sulfonic acid sodium salt dihydrate (PSA,
98%, CAS 10580-19-5), 4-aminoantipyrine (4-AAP, ≥ 99%, CAS
83-07-8) and hydrogen peroxide 30% solution (CAS 7722-84-1) were purchased
from Sigma-Aldrich (St. Louis, Missouri, USA). Trehalose dihydrate
(CAS 6138-23-4) was kindly provided by Hayashibara Co., LDA (Okayama,
Japan). Betaine anhydrous (>97%, CAS 107-43-7) was obtained from
TCI
(Tokyo, Japan) and sucrose (CAS 57-50-1) was purchased from Cmd Chemicals
(Funchal, Portugal).
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