SMCs were isolated from mouse aortas and cell culture lines were established as described previously [28 (link)]. SMCs (2×105) were seeded in 6-well cell culture plates in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and grown to about 80% confluency, then medium was removed and serum-free DMEM/F12 was added to wells. Cells were treated with recombinant PAI-1, PAI-039, or α2-macroglobulin-trypsin complex for 4–24 hrs, after which cells were washed and harvested for analysis. Complexes of human α2-macroglobulin (Sigma-Aldrich, Cat. No. 63013, St. Louis, MO, USA) and trypsin (Sangon Biotech, Shanghai, China) were prepared and treated with soybean trypsin inhibitor (Sangon Biotech) to inactive free trypsin, as described [29 (link), 30 (link)]. In some experiments, SMCs were pre-incubated with a rabbit anti-LRP1 antibody (R2629, 100 µg/mL, kindly provided by Dudley Strickland, PhD, U. Maryland) prior to addition of PAI-1. R2629 does not cross react with any other known LDL receptor family member [31 (link)].
Trypsin
Manufactured by Sangon
Sourced in China
Trypsin is a proteolytic enzyme that is used in laboratory settings to dissociate adherent cells from cell culture surfaces. It functions by cleaving peptide bonds, primarily at the carboxyl side of the amino acids lysine and arginine.
Automatically generated - may contain errors
Lab products found in correlation
Pepsin, by Sangon (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Sangon (4 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Celigo image cytometer, by Revvity (3 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Sangon (2 mentions)
Penicillin streptomycin, by Sangon (2 mentions)
Dulbecco modified eagle medium (dmem), by Sangon (2 mentions)
Dithiothreitol (dtt), by Sangon (2 mentions)
Hemoglobin, by Sangon (2 mentions)
Papain, by Sangon (2 mentions)
D glucose, by Sangon (2 mentions)
Dmem lg, by Thermo Fisher Scientific (2 mentions)
Soybean trypsin inhibitor, by Sangon (1 mentions)
Rnaase, by Tiangen Biotech (1 mentions)
Rpmi 1640 medium, by Sangon (1 mentions)
Dimethyl sulfoxide (dmso), by Sangon (1 mentions)
5702r low speed centrifuge, by Eppendorf (1 mentions)
Cell counting kit 8 cck 8, by Sangon (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
50 protocols using trypsin
1
Isolated SMCs: Regulation by PAI-1 and Complexes
2
Cell Fixation and Propidium Iodide Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were digested into single cells by 0.25% trypsin (Sangon Biotech) and collected into 15-mL centrifuge tubes. Centrifugation at 1000 rpm for 5 min was performed, then we discarded the supernatant. We washed cells twice with 1× PBS buffer and suspended them in 1× PBS buffer by centrifugation at 1000 rpm for 5 min each time. We discarded the supernatant. We suspended cells by 1 mL cold 70% ethyl alcohol and fixed them at 4 °C overnight. We washed cells three times with 1× PBS buffer by centrifugation at 1000 rpm for 5 min each time. We discarded the supernatant and suspended 1 × 106 or 1 × 107 cells with 500 μL 1× PBS buffer including 100 μg/mL propidium iodide (PI) (Biolegend), 50 μg/mL RNAase (TIANGEN), and 0.2% X-Triton 100 (Sangon Biotech), and placed it at 4 °C for 30 min. It was analyzed by flow cytometry.
3
Vanillic Acid and Corticosterone Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vanillic acid (≧98% purity by HPLC) was obtained from Jingzhu Biotechnology Co., Ltd. (Nanjing, China), and corticosterone (purity ≧98%) was purchased from TCI Shanghai (Shanghai, China). RPMI-1640 medium, fetal bovine serum (FBS), 0.25% trypsin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Poly-L-polylysine (PLL) was purchased from Sigma-Aldrich Co. (St. Louis, United States). The commercial kit for measuring the lactate dehydrogenase (LDH) was purchased from Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China).
4
Evaluating Anticancer Compounds in Thyroid Papillary Carcinoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human thyroid papillary carcinoma cell Papillary cells of human thyroid carcinoma BCPAP cell lines (BCPAP) was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Quercetin (CAS No. 117-39-5), luteolin (CAS No. 491-70-3), beta-sitosterol (β-sitosterol) (CAS No. 83-46-5), kaempferol (CAS No. 520-18-3) were purchased from Selleck China company respectively. One thousand six hundred-forty culture medium, dimethyl sulfoxide (DMSO), fetal bovine serum, penicillin streptomycin double antibody, 0.25% trypsin, cell counting kit 8 (CCK8), and DMSO purchased from Sangon Biotech (Shanghai) Co., Ltd. RIPA Lysis Buffer, rabbit derived vascular endothelial growth factor A (VEGFA) antibody, tumor protein p53 (TP53) antibody, transcription factor AP-1 (JUN) antibody, prostaglandin endoperoxidase 2 (PTGS2) antibody, interleukin 6 (IL6) antibody, IL-1B antibody, rabbit derived House keeping protein. (glyceraldehyde-3-phosphate dehydrogenase) antibody and goat anti-rabbit IgG were purchased from Beyotime Biotechnology Co., Ltd. and ThermoFisher Scientific Co., Ltd. respectively. The instrument includes BioRad full-automatic microplate reader, BioRad chemiluminescence gel imaging system, OLYMPUS IX51 inverted microscope, MCO-15AC SANYO CO2 constant temperature incubator, Eppendorf 5702R low-speed centrifuge.
5
Annexin V-FITC/PI Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The percentage of apoptotic cells was determined using an Annexin V-FITC/PI kit (Shanghai Yeasen Biotechnology, Co., Ltd.). Cells were seeded in 6-well plates, at a density of 5×105 cells/well. After incubation for 24 h, the cells were treated with FYGL at a concentration range of 0–500 µg/ml for 24 h and harvested using 0.25% trypsin (Sangon Biotech Co., Ltd.), then the cells were incubated with 5 µl Annexin V-FITC and 10 µl PI working solution for 30 min at 37°C. Finally, the stained cells were analyzed using flow cytometry (Beckman Coulter, Inc.), and the data were analyzed using FlowJo software (version 10.4; BD Biosciences).
6
Alkaloid Compounds Evaluation in Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CKI (total alkaloid concentration of 20.8 mg/mL) was purchased from Shanxi Zhendong Pharmaceutical Co.Ltd. (Shanxi, China), and 5-fluorouracil was ordered from Beijing Solarbio Science Technology Co. Ltd. (Beijing, China). Matrine, oxymatrine, oxysophocarpine and sophoridine were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China; Batch numbers: 110805-200508, 110780-201508, 111652-200301, 110784-201405), and N-methylcytisine was purchased from J&K Scientific Ltd. (Beijing, China). High glucose DMEM, foetal bovine serum (FBS), 0.25% trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulphoxide (DMSO) were obtained from Sangon Biotech Co. (Shanghai, China). Sodium 3-trimethlysilyl [2, 2, 3, 3-d4] propionate (TSP) was purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA). D2O was obtained from Norell (Landisville, Pennsylvania, USA). Primary antibodies against β-actin, MMP2 and Myc were purchased from ProteinTech (Chicago, IL, USA). The caspase-3 antibody was obtained from Cell Signalling Technology, Inc. (Beverly, MA, USA). The REG1A antibody was obtained from Abcam Inc. (Cambridge, MA, USA).
7
Engineered Porous Scaffolds for Cartilage Regeneration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tetraethyl orthosilicate (TEOS), branched polyethyleneimine (PEI, 25 kDa), N-hydroxysuccinimide (NHS), cetyltrimethylammonium chloride solution (CTAC), triethylamine (TEA), and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent (Shanghai, China). VEGF was obtained from PeproTech (Cranbury, NJ, USA). DMEM, trypsin, type Ⅱ collagenase, penicillin/streptomycin, fetal bovine serum, and Alizarin red were purchased from Sangon Biotech (Shanghai, China). Porcine aortic valves were purchased from Songlin Meat Food Co., Ltd. A Cell Counting Kit-8 (CCK-8) and a BCIP/NBT ALP staining kit (Beyotime, Shanghai, China). An alkaline phosphatase assay kit (Jiancheng Bioengineering Institute, Nanjing, China), a calcium content detection kit (MingDian, Shanghai, China), and an RNeasy mini kit were purchased from Qiagen (Duesseldorf, Germany), IL-1, IL-6, IL-10, and TNF-α kit (R&D, Santa Clara, CA, USA). Primary antibodies (GAPDH, OPN, OSX, RunX2, α-SAM, Vimentin, and CD31) and donkey anti-rabbit IgG were procured from Abcam (Cambridge, UK). LipofectamineTM 3000 Transfection Reagent was purchased from Invitrogen (Carlsbad, CA, USA).
8
Pumpkin Seed Protein Extraction and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pumpkin seeds (Cucurbita pepo L.) were provided by Haichuansanxin Food Company (Beitun, China). Alcalase (≥200 U/mg) was purchased by Hefei Bomei Biotechnology Co., Ltd. (Hefei, China). Papain (>200 units/mg) and flavourzyme (30,000 units/g) were purchased from Solarbio Life Science (Beijing, China). Bromelain (3,000–7,000 U/mg), pepsin (1,200 U/g), pancreatin (≥4,000U/g), trypsin (≥50,000U/g), and bile salt and o-Phthalaldehyde (OPA) and dithiothreitol (DTT) were purchased from Sangon Biotech (Shanghai, China). In addition, 8-Anilino-1-naphthalenesulfonic acid (ANS) [≥97% (HPLC)] was purchased from Sigma-Aldrich. Sodium dodecyl sulfate (SDS) and L-Serine (≥BR) were purchased from Sinopharm Chemical Reagent Co., Ltd. Zinc sulfate heptahydrate, sodium hydroxide, nitric acid, n-hexane, ethyl alcohol, and other reagents were of analytical grade and purchased from Chemical Reagent Co., Ltd. (Shanghai, China). Dialysis bags (MD34-500) were purchased from Wuxi Hengkang Medical Technology Co., Ltd.
9
Peptide Synthesis and Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peptides were synthesized and purified by China Peptides Co. Ltd. (Shanghai, People’s Republic of China). Trypsin, hemoglobin, and AA were obtained from Sangon Biotech Co. Ltd. (Shanghai, People’s Republic of China). Bovine serum albumin, thrombin, K2HPO4, KH2PO4, and 6-mercapto-1-hexanol were purchased from Sigma-Aldrich Co. (Shanghai, People’s Republic of China). Hexadecyltrimethylammonium chloride (CTAC) and other reagents were of analytical grade and obtained from the Aladdin Reagent Company (Shanghai, People’s Republic of China). All solutions were prepared freshly with deionized water. The serum samples were supplied by the Anyang Tumor Hospital, People’s Republic of China.
10
Apoptosis Evaluation of ESCC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometer (Millipore, USA) was used to evaluate ESCC cell apoptosis. KYSE-150 and TE-1 infected cells in logarithmic phase of growth were detached by trypsin (Sangon Biotech (Shanghai) Co., Ltd., China) and resuspended in medium. The cell suspension was centrifugated at 1300 rpm for 5 min. D-Hanks precooled at 4°C was carried to wash the cells in absence of supernatant. Bling buffer was applied to additional cell washing and the cells were stained with 10 μl Annexin V-APC, followed by analysis of number of cell apoptosis at different stages by guava InCyte software.
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!