3h proline

Manufactured by PerkinElmer

Sourced in United States

3H-proline is a radioactive amino acid commonly used in research applications. It is a stable isotope-labeled compound that can be detected and quantified through radioactive labeling techniques. This product is intended for use in a variety of research applications, including protein synthesis studies, cell culture experiments, and metabolic tracking.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

L-[2,3-3H]-Proline (3 citations) L-2,3,4,5-3H proline (3 citations)

15 protocols using 3h proline

Quantifying Matrix Synthesis in Cartilage

Check if the same lab product or an alternative is used in the 5 most similar protocols

Synthesis of sGAG and total protein was assessed by supplementing the culture medium with 20 μCi/ml [³⁵S]-sulfate and 10 μCi/ml [³H]-proline (Perkin Elmer, Norwalk, CT), respectively, for 24 h (n = 3/group). Following 4 washes to remove free label, cartilage was separated from bone (in coculture groups) and wet weight (WW) taken for each cartilage and bone sample. Bone tissue was first flash-frozen in liquid nitrogen and crushed immediately into a fine powder. Bone tissue powders and cartilage were digested in 500 μl proteinase K solution (2mg/ml in 50mM Tris-HCl, 1mM CaCl₂, pH8) (Roche, Indianapolis, MN) at 56°C for 16 h. [³⁵S]-sulfate and [³H]-proline incorporation in cartilage digests were measured via liquid scintillation counting (Perkin Elmer) and normalized to wet weights. sGAG content of cartilage tissue digests and spent media was determined using the dimethylmethylene blue (DMMB) dye-binding assay [25 (link)]. Tissue digests were hydrolyzed and dried to assess the total collagen content using the hydroxyproline (OHP) assay [26 (link)] and normalized to wet weights.

Dwivedi G., Flaman L., Alaybeyoglu B., Struglics A., Frank E.H., Chubinskya S., Trippel S.B., Rosen V., Cirit M, & Grodzinsky A.J. (2022). Inflammatory cytokines and mechanical injury induce post-traumatic osteoarthritis-like changes in a human cartilage-bone-synovium microphysiological system. Arthritis Research & Therapy, 24, 198.

+ Open protocol

+ Expand

Quantifying Chondrocyte Collagen Synthesis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Collagen synthesis, analyzed 72 h after cell seeding, was quantified as described previously.^{12 (link)} Briefly, chondrocytes were incubated overnight with 20 µCi per mL ³H-proline (PerkinElmer) and subsequently lysed in 11% acetic acid-H₂O supplemented with 0.25% bovine serum albumin. Proteins were precipitated by the addition of 20% trichloroacetic acid, and the pellet was resuspended in scintillation fluid. Finally, radioactivity was determined by liquid scintillation counting, and the values were normalized to the DNA content.

Stegen S., Loopmans S., Stockmans I., Moermans K., Carmeliet P, & Carmeliet G. (2022). De novo serine synthesis regulates chondrocyte proliferation during bone development and repair. Bone Research, 10, 14.

+ Open protocol

+ Expand

Cardiac Fibroblast Procollagen Synthesis Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cardiac fibroblasts (both control and HF) were treated with the β-agonist isoproterenol/ISO (10 µM), TGF-β (0.5 to 10 ng/mL), MEK1/2 inhibitor PD98059 (100µM, Calbiochem, Billerica, MA) followed by TGF-β (10ng/mL), or MEK1/2 inhibitor U0126 (100µM, Calbiochem, Billerica, MA) followed by TGF-β (10ng/mL) as indicated in the figure legends. Cells were stimulated with ISO or TGF-β for 48 hours. [³H]proline incorporation was measured according to the method of D'Souza et al [10 (link)]. Cells were grown to 80% confluence on 12-well plates, serum starved for 24 hours, and incubated with [³H]proline (1 µCi/well, Perkin Elmer Life Sciences, Shelton, CT, USA) for 48 hr. Cellular protein was precipitated overnight with 20% trichloroacetic acid (TCA) and washed 3 times with 1ml of 5% TCA plus 0.01% proline, then dissolved in 0.2 M NaOH. The activity of [³H]proline was determined by liquid scintillation counting.

Li J., Philip J.L., Xu X., Theccanat T., Razzaque M.A, & Akhter S.A. (2014). β-Arrestins Regulate Human Cardiac Fibroblast Transformation and Collagen Synthesis in Adverse Ventricular Remodeling. Journal of molecular and cellular cardiology, 0, 73-83.

+ Open protocol

+ Expand

Collagen Synthesis in Immortalized HSCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Hepatic stellate cell (HSC) collagen synthesis was analyzed as described earlier (Hodge et al., 2014 (link)). Briefly, human immortalized HSCs (LX2 cell line, a kind gift of Professor Scott Friedman, NY, United States) were serum starved in DMEM containing 5% FBS followed by culture in Ultraculture media overnight at 37°C. In the treatment groups, cells were cultured in 50% Ultraculture media and 50% hAEC-CM, 50% hAEC-EVDM, or 50% PBS with 10 μg EV. In the control groups, HSCs were cultured either in 100% Ultraculture media as a control for CM and EVDM or 50% Ultraculture media and 50% PBS as a control for EV. [³H] Proline (1 μCi, PerkinElmer, Boston, MA, United States) was added to each sample.

Alhomrani M., Correia J., Zavou M., Leaw B., Kuk N., Xu R., Saad M.I., Hodge A., Greening D.W., Lim R, & Sievert W. (2017). The Human Amnion Epithelial Cell Secretome Decreases Hepatic Fibrosis in Mice with Chronic Liver Fibrosis. Frontiers in Pharmacology, 8, 748.

+ Open protocol

+ Expand

Bovine Chondrocyte Isolation and Culture

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immature bovine stifles were from Research 87 (Marlborough, MA). High glucose Dulbecco’s modified Eagle’s medium (DMEM) was from HyClone (Logan, UT). Non-essential amino acids (NEAA) and proteinase K were from Life Technologies (Carlsbad, CA). N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES), gentamicin and phosphate buffered saline (PBS) were from Mediatech (Manassas, VA). Insulin-transferrin-selenous acid premix (ITS+) was from BD Biosciences (Franklin Lakes, NJ). L-ascorbic acid 2-phosphate, proline, ammonium acetate, 1,9-dimethyl-methylene blue (DMMB), shark chondroitin sulfate, sodium nitrite, bisbenzimide (Hoechst 33258), and calf thymus DNA were from Sigma (St. Louis, MO). Sulfanilamide reagent and naphthylethylenediamine dihydrochloride solution were from Ricca Chemical (Arlington, TX). The CytoTox-ONE^™ Homogeneous Membrane Integrity assay kit was from Promega (Madison, WI). ³H-proline and ³⁵S-sodium sulfate were from Perkin Elmer (Waltham, MA). Recombinant human leptin was from Shenandoah Biotechnology (Warwick, PA), recombinant human visfatin was from Novus Biologicals (Littleton, CO), and recombinant human adiponectin and resistin were from Biovendor (Candler, NC).

Nishimuta J.F, & Levenston M.E. (2015). Meniscus is more susceptible than cartilage to catabolic and anti-anabolic effects of adipokines. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society, 23(9), 1551-1562.

+ Open protocol

+ Expand

Angiotensin II-Induced Cardiac Fibroblast Proliferation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cardiac fibroblasts (CFs) were pre‐incubated with Ang II for 4 hrs before the proliferation assay. The ³H‐proline incorporation assay was performed as described previously 15. In brief, 5 × 10³ CF cells per well in serum‐free medium and incubated overnight. EGCG, SP600125, JNK1 siRNA and DMSO were subsequently added to the plate. Proline uptake was measured by adding 500 nCi/ml [3H]‐proline (Perkin Elmer, Boston, MA, USA). MicroScint‐20 (50 μl) was added, and the plate was read using TopCount (Packard Instrument).

Lin C., Chang H., Wang B, & Shyu K. (2016). Suppressive effect of epigallocatechin‐3‐O‐gallate on endoglin molecular regulation in myocardial fibrosis in vitro and in vivo. Journal of Cellular and Molecular Medicine, 20(11), 2045-2055.

+ Open protocol

+ Expand

Quantification of Macromolecular Synthesis in Chondrocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols

Collagen, proteoglycan or total protein synthesis was quantified in vitro, by incubation of cultured chondrocytes with 20 uCi/ml ³H-proline (PerkinElmer), ³⁵S-sulphate (PerkinElmer) or ³⁵S-methionine (MP Biomedicals), respectively, as described before^{36 ,37}. Briefly, after overnight labelling, cells were lysed in extraction buffer (11% acetic acid H₂O with 0.25% BSA) to quantify collagen and total protein synthesis and proteins were precipitated by the addition of 20% trichloroacetic acid. For the analysis of proteoglycan synthesis, cells were lysed in 0.2 M NaOH and proteoglycans were precipitated with 1% cetylpyridinium chloride. Radioactivity was determined by liquid scintillation counting, and normalized for DNA content.

Stegen S., Laperre K., Eelen G., Rinaldi G., Fraisl P., Torrekens S., Van Looveren R., Loopmans S., Bultynck G., Vinckier S., Meersman F., Maxwell P.H., Rai J., Weis M., Eyre D.R., Ghesquière B., Fendt S.M., Carmeliet P, & Carmeliet G. (2019). HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes. Nature, 565(7740), 511-515.

+ Open protocol

+ Expand

Mast Cell Differentiation and Activation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dulbecco's Modified Eagle's Medium (DMEM), Iscove’s modified Dulbecco’s medium (IMDM), fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from Life Technologies (Gibco). L-glutamine, ascorbic acid, 1-thioglycerol, HEPES buffer, nonessential amino acids and sodium pyruvate were purchased from Sigma. Human stem cell factor (SCF), interleukin 6 (IL-6) and interleukin 3 (IL-3) were obtained from Peprotech. Recombinant Human MIF (Z03159-50) was obtained from Genscript. 6-well plates and 10-cm ultra-low-attachment dishes were purchased from Corning. Anti-chymase (MA5-11717) and anti-tryptase (MA5-11711) antibodies were purchased from Thermo Scientific Pierce Antibodies. Human IgE, anti-IgE antibody (ab75673) and anti-MIF antibody (ab7207) were bought from Abcam. Phycoerytrin (PE)-labeled anti-CD34 antibody (HPCA-2) and PE-IgG isotype control were purchased from Becton Dickinson (BD). Anti-human FcεRI-FITC and anti-human CD117-PE were purchased from eBioscience. SPI Bio histamine EIA kit was obtained from Bertin Pharma. [3H]-proline was from Perkin-Elmer.

Ningyan G., Xu Y., Hongfei S., Jingjing C, & Min C. (2015). The Role of Macrophage Migration Inhibitory Factor in Mast Cell-Stimulated Fibroblast Proliferation and Collagen Production. PLoS ONE, 10(3), e0122482.

+ Open protocol

+ Expand

Radiolabeled Amino Acids for Protein Synthesis

Check if the same lab product or an alternative is used in the 5 most similar protocols

³H-Leucine (leucine, l-[4,5-³H]), ³H-phenylalanine (phenylalanine, l-[2,3,4,5,6-³H]), ³H-tryptophan (tryptophan, l-[5-3H(N)]), ³H-methionine (l-[methyl-³H]-methionine), ³H-tyrosine (l-[ring-3,5-³H]-tyrosine), ³H-arginine (arginine monohydrochloride l-[2,3,4-³H]-arginine), ³H-lysine (l-[4,5-³H(N)]-lysine), ³H-proline (l-[2,3,4,5-³H]-proline), ³H-serine (l-[³H(G)]-serine), ³H-glycine (glycine, [2-³H]-glycine), ³H-glutamine (l-[3,4-³H(N)]-glutamine), and ³H-glutamic acid (l-[3,4-³H]-glutamic acid) are from PerkinElmer (Waltham, MA).

Bandyopadhyay U., Todorova P., Pavlova N.N., Tada Y., Thompson C.B., Finley L.W, & Overholtzer M. (2022). Leucine retention in lysosomes is regulated by starvation. Proceedings of the National Academy of Sciences of the United States of America, 119(6), e2114912119.

+ Open protocol

+ Expand

Extracellular Matrix Biosynthesis Characterization

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Synthesis of total protein and sulfated glycosaminoglycans (sGAG) was measured by incorporation of ³H-proline (5 μCi/mL) and ³⁵S-sulfate (20 μCi/mL), respectively (Perkin-Elmer, Norwalk, CT). Cell proliferation was also measured by incorporation of ³H-thymidine (1 μCi/mL, Perkin-Elmer), but in different experiments than those measuring ³H-proline incorporation. All radiolabels were added to the culture medium for 24 hours. When terminated, explants were washed in 1x PBS, weighed and digested with proteinase K (5mg/ml) (Roche, Indianapolis, MN) for 16-18h. Radiolabel incorporation was measured using a liquid scintillation counter (Perkin-Elmer). sGAG content and total collagen content were measured from the digested explants using the dimethylmethylene blue (DMMB)^{39 (link)} and hydroxyproline (OHP) assays,^{40 (link)} respectively, as described previously. All biochemical analyses were normalized to DNA content, which was measured using the PicoGreen dye-binding assay.^{41 (link)} Biosynthesis and biochemical analyses were performed every other day in culture (days 1, 3, 5, and 7).

Connizzo B.K, & Grodzinsky A.J. (2018). Release of Pro-Inflammatory Cytokines from Muscle and Bone Causes Tenocyte Death in a Novel Rotator Cuff In Vitro Explant Culture Model. Connective tissue research, 59(5), 423-436.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!