Tube formation assay was carried out as described before [66 (link)]. VR-EPCs with a density of 3 × 104 cells/cm2 were seeded on the surfaces of sterilized Ti, TNrs, and Fe3O4-TNrs or into the plates wells without any sheets. According to different experimental purposes, VR-EPCs were treated with 1 mT 15 Hz SEMF or 1 μM Cyclosporin A (CsA, CaN inhibitor) (Selleck, USA), then the cells were digested by using trypsin (Boster, Wuhan, China). 4°C-cooled Matrigel (BD Company, USA) was put into the bottom of each well of 48-well plates (200 μL/well), and the plates were placed in a 37 °C environment for 30 min to solidify the Matrigel. VR-EPCs were seeded on the solidified Matrigel at a density of 2 × 104 cells/well and cultured in the cell incubator. 4 h later, the automatic cell imaging system (EVOS FL Auto, Life Technologies, USA) was used to collect the images of each well, and Image J software was used to analyze the images.
Trypsin
Manufactured by Boster Bio
Sourced in China, United States
Trypsin is a proteolytic enzyme that is used for the dissociation and disaggregation of adherent cells in cell culture applications. It functions by cleaving peptide bonds at the carboxyl side of lysine and arginine residues, allowing cells to be detached from the culture surface or each other.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Collagenase type 2, by Boster Bio (3 mentions)
Penicillin streptomycin, by Boster Bio (2 mentions)
Mmp13 18165 1 ap, by Proteintech (2 mentions)
Safranin o solution, by Solarbio (2 mentions)
Isobutylmethylxanthine ibmx i7018, by Merck Group (2 mentions)
Mitotracker red cmxros, by Solarbio (2 mentions)
Evos fl auto, by Thermo Fisher Scientific (1 mentions)
Sodium taurocholate, by Merck Group (1 mentions)
Penicillin streptomycin, by Sartorius (1 mentions)
Rpmi 1640 medium, by Sartorius (1 mentions)
Facscalibur, by BD (1 mentions)
Ficoll paque, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Pi rnase staining solution, by Solarbio (1 mentions)
Rapamycin rapa, by Selleck Chemicals (1 mentions)
Yap 14074, by Cell Signaling Technology (1 mentions)
P yap, by Cell Signaling Technology (1 mentions)
A11691, by ABclonal (1 mentions)
Ip lysis buffer p0013, by Beyotime (1 mentions)
16 protocols using trypsin
1
Tube Formation Assay of VR-EPCs
2
Propagation and Purification of Cryptosporidium parvum
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HCT-8 cells were preserved and propagated in our laboratory. HCT-8 cells were cultured with RPMI 1640 medium (Biological Industries, Israel) containing 10% FBS and 1% penicillin streptomycin (Biological Industries, Israel) at 37 °C in 5% CO2. When grown to 70–80% confluence, the cells were subjected to passaging or plating.
Cryptosporidium parvum (gene subtype: IIa A15G2R1) was preserved and propagated in our laboratory. Cryptosporidium oocysts, originally obtained from our laboratory, were used to infect 5-day-old Holstein calves (1×108/animal). Following the onset of oocyst shedding, feces were collected daily, mixed with an equal volume of 5% potassium dichromate and stored at 4 °C. Oocyst isolation was through discontinuous sucrose gradients according to previous methods [33 (link)]. For purification of sporozoites, oocysts were resuspended in 0.8% sodium taurocholate (Sigma, MO, USA) with 2.5% trypsin (BOSTER, Wuhan, China), then incubated at 37 °C for 30 min, washed three times in PBS and resuspended in RPMI 1640 medium with 2% FBS and 1% penicillin-streptomycin. The excystation rate of sporozoites is about 70% using a cell counting plate, and the number of oocysts required is calculated as a ratio of 1:2 of sporozoites to HCT-8 cells.
Cryptosporidium parvum (gene subtype: IIa A15G2R1) was preserved and propagated in our laboratory. Cryptosporidium oocysts, originally obtained from our laboratory, were used to infect 5-day-old Holstein calves (1×108/animal). Following the onset of oocyst shedding, feces were collected daily, mixed with an equal volume of 5% potassium dichromate and stored at 4 °C. Oocyst isolation was through discontinuous sucrose gradients according to previous methods [33 (link)]. For purification of sporozoites, oocysts were resuspended in 0.8% sodium taurocholate (Sigma, MO, USA) with 2.5% trypsin (BOSTER, Wuhan, China), then incubated at 37 °C for 30 min, washed three times in PBS and resuspended in RPMI 1640 medium with 2% FBS and 1% penicillin-streptomycin. The excystation rate of sporozoites is about 70% using a cell counting plate, and the number of oocysts required is calculated as a ratio of 1:2 of sporozoites to HCT-8 cells.
3
Isolation and Characterization of Menstrual Stem Cells
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Approximately 5 ml of menstrual blood was collected from healthy female subjects with normal menstrual cycles. The menstrual blood was transferred to PBS containing amphotericin B (Sigma-Aldrich, US) and penicillin/streptomycin (1%) (HyClone, US). After incubation at 4°C for 24 hours, the sample was centrifuged at 1600 g for 10 minutes at 4°C, and the supernatant was subjected to microbiological examination. Mononuclear cells were separated by Ficoll-Paque (Thermo Fisher Scientific, USA) density gradient centrifugation and washed twice with PBS. Purified monocytes were cultured using Chang's medium (Laboserv, Germany). After 4-6 days of culture, cells were digested with trypsin (Boster, China) for passage. The 3rd-6th passage cells were taken to carry out the experiment. For the identification of MenSCs, the expression levels of stem cell positive markers CD44, CD90, and CD105 and negative markers CD34 and CD45 (Thermo Fisher Scientific, USA) were detected by flow cytometry (BD FACSCalibur, USA).
4
Flow Cytometry Analysis of Pyroptosis
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Adherent cells were digested with 0.25% trypsin (Boster Biological Technology, CA, USA). Pyroptosis Wash Buffer was diluted 1:10 with double-distilled water. FLICA was diluted with 50 µL of DMSO, and 200 µL of PBS was added to dilute FLICA 1:5. Diluted FLICA was added to each group of cells at a ratio of 1:30 and incubated for 1 h. The cells were washed three times and analyzed by flow cytometry (Agilent, CA, USA).
5
Cell Cycle Arrest Assay for Cancer Cells
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The cell cycle experiment is a traditional method used to study the effect of drugs on the cell cycle [40 (link),41 (link)]. We used FBS-free DMEM treatment, cultured at 37 °C in a 5% CO2 constant temperature and humidified incubator for 24 h, so that the cancer cells were blocked in the G0/G1 period. Briefly, Smmc-7721 cells (106 per well) were incubated with Medium 1640. Cells were collected with trypsin (BOSTER, Shanghai, China) and washed twice with PBS. The resuspended cells were slowly added to precooled 70% ethanol and stored at −20 °C, and at 4 °C for long-term storage. The resuspended cells were washed twice with PBS, added to PI/RNase staining solution (Solarbio, Beijing, China), and the cells were stained in the dark for 30–60 min. Cell cycle distribution was confirmed by measuring the PI fluorescence signal with a BD-C5 flowmeter (measured in 30,000 cells).
6
Chondrocyte Inflammatory and Catabolic Regulation
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The list of regents was obtained commercially: Recombinant Rat interleukin 1 beta (IL-1β) (501-RL-010) was acquired from R&D Systems (Minneapolis, MN, USA). Rapamycin (Rapa) (S1039) was acquired from Selleck (Houston, USA) and diluted in DMSO. Antibodies against COX2 (12882; 1:1000), YAP (14074; 1:1000), P-P65 (3033; 1:1000), P-YAP (13008; 1:1000) and P65 (8242; 1:1000) were gained from Cell Signaling Technology Inc. (Beverly, USA). Antibodies against MMP13 (18165-1-AP; 1:1000), COL2A1 (28459-1-AP; 1:800), GAPDH (10494-1-AP; 1:5000), P-RIPK1 (66854-1-Ig; 1:2000) and β-actin (CL594-66009; 1:5000) were acquired from Proteintech Group (Wuhan, China). Antibodies against iNOS (A0312; 1:1000) and aggrecan (A11691; 1:1000) were acquired from Abclonal (Wuhan, China). Antibodies against P62 (GB11531-100; 1:1000) was acquired from Servicebio (Wuhan, China). Antibodies against MMP3 (BM4074; 1:500), FITC-conjugate goat anti-mouse and anti-rabbit secondary antibodies, type II collagenase and trypsin were purchased from Boster (Wuhan, China). IP lysis buffer (p0013) was provided by Beyotime (Shanghai, China).
7
In Vitro Evaluation of Antioxidant and Antiapoptotic Effects of Traditional Chinese Medicine
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All herbal medicines were provided by Guangyuan Hospital of Traditional Chinese Medicine (Guangyuan, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM) were purchased from the Gibco Co. (Grand Island, NY, United States). BCA protein assay reagents, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) preparation kit, penicillin-streptomycin mixture, trypsin, phosphate buffer saline (PBS) and cell counting kit-8 (CCK-8) were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). Annexin V-FITC/PI assay kit was purchased from Multisciences (Lianke) Biotechnology Corporate Limited (Hangzhou, China). H2O2 was purchased from Chengdu Chron Chemicals Co. Ltd. (Chengdu, China). Primary antibodies for PI3K, phosphorylation-PI3K (p-PI3K), AKT, phosphorylation-AKT (p-AKT), Nrf2, Bcl-2, Bax and cleaved-caspase-3 (C- caspase-3) were obtained from the ImmunoWay Biotechnology Co. (Suzhou, China). The assay kits for MDA, SOD, CAT, and GSH-PX were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Ultrapure water purified by Millipore Ultra-pure Water Purifier (Millipore, Milford, MA, United States) was used. Other reagents were all of analytical grade.
8
Chondrocyte Senescence and Apoptosis
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FKA (T3S0737, purity: 99.41%) and 3-Methyladenine (3 MA) were acquired from TOPSCIENCE (Shanghai, China), and the mouse IL-1β cytokine was obtained from the R&D system (501-RL-010, United States). Safranin O solution were provided by Solarbio (Beijing, China). The primary antibodies for iNOS, COX2, Col2, Aggrecan, Sox9, and immunofluorescence secondary antibodies were acquired from Abcam (Shanghai, China). Atg12, Beclin, LC3I/II, P21, P16, Bax, Bcl2, GAPDH, and antibodies for all pathways were purchased from CST (Beverly, MA, United States). Corresponding primary antibodies for MMP3/13 and Nrf2/HO-1/NQO1 were supplied by the Proteintech Group (Wuhan, Hubei, China). ADAMTS5 primary antibody, secondary antibody, phosphate-buffered saline (PBS), trypsin, collagenase type II, the CCK8 assay kit, bovine serum albumin (BSA), and protein extraction kit were ordered and acquired from Boster Biological Technology (Wuhan, Hubei, China). RFP-GFP-LC3-adenovirus was purchased from Hanbio (Shanghai, China). The Senescence β-galactosidase staining kit and the Annexin V-FITC apoptosis detection kit were supplied by Beyotime (Shanghai, China).
9
Isolation of Neonatal Fibroblasts from GFP Mice
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Fibroblasts were isolated from GFP transgenic neonatal mice as previously described by Cheng et al.59 (link). First, skin tissue obtained from neonatal mice was washed with PBS three times and then immersed in 0.5 mg/mL dispase II (Sigma, USA) at 4 °C overnight to separate the epidermis and dermis. Second, after the epidermis was removed, the dermis was minced and digested in 1 mL of 0.25 mg/mL trypsin (Boster, China) for 10 min. Then, 3 mL of Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) was added to discontinue the digestion, and the mixture solution was centrifuged at 1000 rpm for 6 min. Finally, the cells were collected and incubated in DMEM containing 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) in a 5% CO2 incubator at 37 °C.
10
Alantolactone Modulates Inflammatory Response
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Alantolactone (HY-N0038) was obtained from MedChem Express (MCE). Recombinant mouse IL-1β cytokine was provided from R&D Systems (501-RL-010, United States). Solarbio supplied safranin O liquor (Beijing, China). Primary antibodies applied in this research were: anti-iNOS, anti-MMP-13 which were acquired from Abcam (Shanghai, China), anti-COX2, anti-LC3Ⅱ/Ⅰ, anti-P-STAT3, anti-P-P65, anti-P-IκB, anti-P/T-mTOR which were purchased from CST (Beverly, MA, United States), anti-STAT3, anti-MMP-1, anti-MMP-3, anti-ATG5, anti-P62, anti-P65, anti-IκB, anti-P/T-PI3K, anti-P/T-AKT, anti-GAPDH which were afforded from Proteintech Group (Wuhan, Hubei, China) and anti-ADAMTS5 which was supplied from Boster Biological Technology (Wuhan, Hubei, China). Secondary antibodies, Cy3 and FITC Conjugated AffiniPure Goat Anti-Rabbit IgG, DAPI staining solution, collagenase type II and trypsin were got from Boster Biological Technology (Wuhan, Hubei, China). HanBio Inc. (Shanghai, China) served the mRFP-GFP-LC3 adenoviral vectors.
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