L proline

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

L-proline is an amino acid that can be used as a laboratory reagent. It is a naturally occurring compound that is a component of many proteins. L-proline is commonly used in various biochemical and cell culture applications.

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Close spelling variants of this product

Proline (15 citations) 4-oxo-L-proline hydrobromide (3 citations) Trans-4-hydroxy-L-proline (2 citations)

39 protocols using l proline

Quantification of Targeted Metabolites by UHPLC-QTOF-MS

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Pure standards of five targeted metabolites, namely L-proline, spermine, acetylcarnitine, L-tryptophan and lysophosphatidylcholine (LPC, 18:2) were purchased, as follows:

L-proline (from Amino Acid Standard H product #20088, Thermo Scientific, Waltham, MA, USA). MW = 115.

spermine (>97% product S3256, Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA). MW = 202.

o-acetyl-L-carnitine hydrochloride (J6153606; Alfa Aesar by Thermo Scientific, Waltham, MA, USA). MW = 203.

L-tryptophan (>98.5% (T) (HPLC) (T0541;TCI Chemicals, Portland, OR, USA). MW = 204.

lyso L-α-Lysophosphatidylcholine-LPC(18:2) from bovine brain (CAS nr. 9008-30-4) Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA. MW = 519.

Other reagents such as HPLC-grade methanol, formic acid and acetonitrile were purchased from Sigma-Aldrich Chemie GmbH (St. Louis, MO, USA) and Thermo Fisher Scientific (Waltham, MA, USA). Ultrahigh-purity water was prepared by a Millipore-Q Water Purification System (Millipore, Darmstadt, Germany).
The instruments used in this study included a vortex mixer, Minicentrifuge Eppendorf (Thermo Fisher Scientific, Waltham, MA, USA) and UHPLC-QTOF-MS (Bruker GmbH, Bremen, Germany).

Nitusca D., Socaciu C., Socaciu A.I., Sirbu I.O., Bardan R., Cumpanas A.A., Seclaman E, & Marian C. (2023). Potential Diagnostic Biomarker Detection for Prostate Cancer Using Untargeted and Targeted Metabolomic Profiling. Current Issues in Molecular Biology, 45(6), 5036-5051.

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Cultivation of Three-Dimensional Bone-Like Constructs

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The media and reagents used in this experiment have all been previously described.^{5 (link)} All solutions and media were prepared and stored at 4°C and were warmed to 37°C in a heated bead bath before use. Briefly, growth medium (GM) consisted of 78% Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY), with 20% fetal bovine serum (FBS; Gibco), 2% antibiotic–antimycotic (ABAM; Invitrogen, Grand Island, NY), 6 ng/mL basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ), 0.13 mg/mL ascorbic acid-2-phosphotase (Sigma-Aldrich, St. Louis, MO), and 0.05 mg/mL l-proline (Sigma-Aldrich); differentiation medium (DM) consisted of 91% DMEM, 7% horse serum albumin (HS; Gibco), 2% ABAM, 0.13 mg/mL asc-2-phos, 0.05 mg/mL l-proline, and 2 ng/mL transforming growth factor-beta (TGF-β; PeproTech). For the culture of bone-like constructs, 10⁻⁸ M DEXamethasone (DEX; Sigma-Aldrich) was added to GM and DM.^{9 (link)}Construct dishes were prepared as described previously^{5 (link)} to house and constrain the formed three-dimensional constructs. Briefly, 100-mm diameter cell culture plates were filled with 12 mL Sylgard (Dow Chemical Corp., Midland, MI; type 184 silicon elastomer) and allowed to cure for 3 weeks at room temperature. Before use, plates were decontaminated with UV light (wavelength 253.7 nm) for 60 min and rinsed with 70% EtOH and DPBS.

Mahalingam V., Wojtys E.M., Wellik D.M., Arruda E.M, & Larkin L.M. (2016). Fresh and Frozen Tissue-Engineered Three-Dimensional Bone–Ligament–Bone Constructs for Sheep Anterior Cruciate Ligament Repair Following a 2-Year Implantation. BioResearch Open Access, 5(1), 289-298.

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Chondrocyte Differentiation Protocol

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Media were prepared, as previously described, with slight modifications.^{15 (link),17 (link)} Growth medium (GM) consisted of Dulbecco's modified Eagle's medium (DMEM; Gibco, Cat# 10565-042) supplemented with 20% Fetal Bovine Serum (FBS; Gibco, Cat# 10437-028), 2% antibiotic-antimycotic (Sigma; Cat# A9909), 600 ng/mL bFGF, and 40 ng/mL dexamethasone. Differentiation medium (DM) consisted of DMEM supplemented with 7% Horse Serum Albumin (HS; Gibco, Cat# 16050-122) and 2% antibiotic-antimycotic, plus the addition of 0.13 mg/mL ascorbic acid-2 phosphate, 0.05 mg/mL L-proline, 400 ng/mL dexamethasone, and 2 ng/mL transforming growth factor-beta (TGF-β; Peprotech, Cat# 100-21).

Smietana M.J., Moncada-Larrotiz P., Arruda E.M., Bedi A, & Larkin L.M. (2017). Tissue-Engineered Tendon for Enthesis Regeneration in a Rat Rotator Cuff Model. BioResearch Open Access, 6(1), 47-57.

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Chondrogenic Pellet Culture Protocol

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Pellet culture was performed by spinning down 3×10⁵ cultured cells in each 15ml conical tube (BD Falcon) which were then grown in serum-free DMEM containing an insulin-transferrin-selenious acid mix (ITS) (Becton-Dickinson), 50 μg/ml L-ascorbic acid 2-phosphate (WAKO), 100 μg/ml sodium pyruvate, 40 μg/ml L-proline (both from Invitrogen, Life Technologies), 0.1 μM dexamethasone (Sigma-Aldrich) and 10 ng/ml transforming growth factor β1 (TGF-β1; Peprotech) (13 (link)). Three weeks later, pellets were fixed in 10% formalin, dehydrated in ethanol, and embedded in paraffin. Sections (5 μm thick) were rehydrated and stained with Alcian blue and nuclear fast red for the detection of sulfated glycosaminoglycans and nuclei, respectively.

Chen W.C., Baily J.E., Corselli M., Diaz M., Sun B., Xiang G., Gray G.A., Huard J, & Péault B. (2015). Human Myocardial Pericytes: Multipotent Mesodermal Precursors Exhibiting Cardiac Specificity. Stem cells (Dayton, Ohio), 33(2), 557-573.

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Chondrogenic Differentiation of hBMSCs

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For chondrogenic differentiation, hBMSCs were cultured as micro-mass. Cells were harvested and dotted in the center of each well of 24-well plates at a density of 1 × 10⁵ cells/10 μL. After allowing the cells to adhere to the surface of the plates for 2 h at 37 °C, chondrogenic differentiation medium composed of DMEM-HG containing 1% antibiotic-antimycotic solution, 1% Insulin Transferrin Selenium-A (ITS) (Invitrogen, Carlsbad, CA, USA), 1 μM dexamethasone, 10 μM L-proline, 50 μg/mL ascorbic acid (Invitrogen), and 10 ng/mL TGF-β3 (R&D Systems, Minneapolis, MN, USA) was gently added, and the culture was maintained for 14 days. The chondrogenic differentiation medium was replenished every 2 days. Micro-mass pellets were then fixed in 4% paraformaldehyde for 2 days and then stained with alcian blue to detect proteoglycans.

Kim Y.J., Park K.H., Lee K.M., Chun Y.M, & Lee J.W. (2022). Deubiquitinating Enzyme USP7 Is Required for Self-Renewal and Multipotency of Human Bone Marrow-Derived Mesenchymal Stromal Cells. International Journal of Molecular Sciences, 23(15), 8674.

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Immortalized Human MSCs on 3D Scaffolds

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Human mesenchymal stem cells (MSCs) were immortalised using a previously published protocol [33] . Both 3D nanofibrous and smooth-surface scaffolds were sterilised by soaking in 70% ethanol for 30 min, washed three times in PBS and twice in expansion medium (Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, 1% non-essential amino acids, 1% L-glutamine and 1% antibiotic/antimycotic solution). 4 x 10 6 cells were manually seeded onto each scaffold. Three hours after seeding, either the expansion medium, chondrogenic differentiation medium (serum-free expansion medium supplemented with 50 µg/ml L-ascorbic acid phosphate, 40 µg/ml L-proline, 1% ITS+, 1 mM pyruvate and 10 ng/ml TGF-β1, all from Invitrogen, UK) or osteogenic differentiation medium (αMEM supplemented with 10% FBS, 1% L-glutamine and 1% antibiotic/antimycotic solution, 10 mM β-glycerophosphate and 100 nM dexamethasone) was added. The medium was changed twice a week.

Prasopthum A., Shakesheff K.M, & Yang J. (2018). Direct three-dimensional printing of polymeric scaffolds with nanofibrous topography. Biofabrication, 10(2).

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Chondrocyte Culture Methodology

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Dulbecco’s modified Eagle’s medium (DMEM) was from Cellgro (Manassas, VA). HEPES, 100× non-essential amino acids (NEAA), and 100× insulin-transferrin-selenium (ITS) were purchased from Gibco (Carlsbad, CA). Ascorbic acid and l-proline were from Fisher Bioreagents (Pittsburgh, PA). Human recombinant IL-1α and human recombinant IL-1Ra were from PeproTech (Rocky Hill, NJ). Radiolabeled ³⁵S-sulfate was from PerkinElmer (Waltham, MA). Proteinase K was purchased from Roche Diagnostics (Risch-Rotkreuz, Switzerland). Dermal punches were purchased from Moore Medical (Farmington, CT). Tissue culture well plates were from Cellgro (Manassas, VA). Additional reagents were from Sigma-Aldrich (St. Louis, MO) where not otherwise noted.

Mehta S., Akhtar S., Porter R.M., Önnerfjord P, & Bajpayee A.G. (2019). Interleukin-1 receptor antagonist (IL-1Ra) is more effective in suppressing cytokine-induced catabolism in cartilage-synovium co-culture than in cartilage monoculture. Arthritis Research & Therapy, 21, 238.

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Chondrocyte Differentiation Induction Protocol

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Human recombinant IL-1α and human recombinant IGF-1 were purchased from PeproTech (Rocky Hill, NJ). Dulbecco’s modification of Eagle’s medium (DMEM) was purchased from Corning (Corning, NY). Trypsin-EDTA phenol red, HEPES buffer, non-essential amino acids (NEAA), and penicillin streptomycin antibiotic-antimycotic (PSA) were purchased from Gibco (Carlsbad, CA). Ascorbic acid (AA) and L-proline were from Fisher Bioreagents (Pittsburgh, PA). Proteinase-K was purchased from Roche Diagnostics (Risch-Rotkreuz, Switzerland). NHS-PEG_2-Maleimide and Protease Inhibitor Mini Tablets were from Thermo Scientific Pierce (Rockford, IL). Propidium iodide (PI) was obtained from Thermofisher Acros Organics (Geel, Belgium). Tris-HCl buffer was purchased from Invitrogen (Grand Island, NY). Chondroitinase ABC, Resazurin sodium salt, Griess reagent, fluorescein diacetate (FDA), and other salts and reagents were purchased from Sigma (St. Louis, MO).

Vedadghavami A., Hakim B., He T, & Bajpayee A.G. (2022). Cationic peptide carriers enable long-term delivery of insulin-like growth factor-1 to suppress osteoarthritis-induced matrix degradation. Arthritis Research & Therapy, 24, 172.

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Protease Inhibitor Mini Tablet Assay

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Protease Inhibitor Mini Tablets were purchased from Thermo Scientific Pierce (Rockford, IL). Proteinase-K was purchased from Roche Diagnostics (Risch-Rotkreuz, Switzerland). Dulbecco’s Modification of Eagle’s Medium (DMEM) was from Cellgro (Manassas, VA). HEPES, Insulin-transferrin-selenium (ITS), trypsin-EDTA phenol red and non-essential amino acids (NEAA) were purchased from Gibco (Carlsbad, CA). Ascorbic acid and L-proline were from Fisher Bioreagents (Pittsburgh, PA). Sulfur-35 radionuclide was obtained from PerkinElmer (Waltham, MA). Propidium Iodide (PI) was obtained from Thermofisher Acros Organics (Geel, Belgium). Fluorescein Diacetate (FDA), Nonfat-dried bovine milk and other salts and reagents were purchased from Sigma (St. Louis, MO).

Vedadghavami A., Wagner E.K., Mehta S., He T., Zhang C, & Bajpayee A.G. (2018). Cartilage Penetrating Cationic Peptide Carriers for Applications in Drug Delivery to Avascular Negatively Charged Tissues. Acta biomaterialia, 93, 258-269.

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Production and Purification of SDF1-ELP

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The pET25B+ vector with SDF1-ELP was retransformed in E. coli (BL21 Star DE3), which was obtained from Invitrogen by Life Technologies. One bacteria colony was picked for an overnight culture in 5 ml LB medium containing 25μg/mL carbenicillin. The overnight culture was used to inoculate 500 mL of terrific broth supplemented with 100 mM of L-proline (Fisher Scientific) and with 25μg/mL carbenicillin. The culture was monitored until it reached an optical density at 600 nm of about 0.6, after which 0.5 mM of IPTG (Sigma) was added to induce the protein. The culture was left overnight. The next day, the culture was centrifuged at 3000 x g, and the pellet diluted with 40 ml of PBS and the suspension sonicated twice on ice for 9 min in cycles of 10 s on and 20 s off. Poly(ethyleneimine) solution (Sigma Aldrich) was added to a final concentration of 0.5% w/v to remove residual DNA, and after centrifuging, SDF1-ELP protein transition to nanoparticles was induced with the addition of 1M NaCl and warming to about 40°C.

Yeboah A., Cohen R.I., Faulknor R., Schloss R., Yarmush M.L, & Berthiaume F. (2016). The development and characterization of SDF1α-elastin-like-peptide nanoparticles for wound healing. Journal of controlled release : official journal of the Controlled Release Society, 232, 238-247.

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