To investigate the effect of inflammation on P2X7R expression in PDLSCs, we used TNF-α (10 ng/mL) and IL-1β (5 ng/mL; both from Novoprotein Scientific Inc., Shanghai, China) to establish an in vitro inflammatory microenvironment according to previous publications69 (link),70 (link). mRNA and protein expression of P2X7R in PDLSCs under normal and osteogenic conditions was determined by quantitative real-time polymerase chain reaction (qRT-PCR) (see Section qRT-PCR) and western blot analysis (see Section Western blot analysis), respectively. Cells cultivated in parallel in a normal microenvironment were used as the control.
Il 1β
Manufactured by Novoprotein
Sourced in China
IL-1β is a recombinant human interleukin-1 beta protein. It is a pro-inflammatory cytokine that plays a central role in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Novoprotein (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
α btx, by Bio-Techne (1 mentions)
Recombinant human il 8, by Novoprotein (1 mentions)
Interleukin 6 (il 6), by Novoprotein (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Blood cell separator, by Fresenius (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Aim 5 cell culture medium, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Multifunction microplate reader, by Agilent Technologies (1 mentions)
Bilobalide, by Novoprotein (1 mentions)
Cck 8, by Apexbio (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
9 protocols using il 1β
1
Inflammation Modulates P2X7R Expression in PDLSCs
2
Chondrocyte Degradation Mechanisms
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The rabbit immortalized mandibular condylar chondrocyte (IMCC) cell line used in this study was purchased from the Department of Oral Biology of the School of Stomatology of the Fourth Military Medical University (China). The IMCCs were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) supplemented with 10% FBS (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) and penicillin/streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) at 37°C in a 5% CO2 atmosphere.
A total of 1 × 105 cell/mL IMCCs were plated on 6-well plates and treated with 10 ng/mL IL-1β (Novoprotein, Shanghai, China) to induce chondrocyte degradation. Samples were collected at different time points (1, 2, 3, 6, 12, and 24 h), and the expression levels of ECM-related genes were analyzed by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), including aggrecan, type II collagen, ADAMTS-4, ADAMTS-5, and MMP-9.
A total of 1 × 105 cell/mL IMCCs were plated on 6-well plates and treated with 10 ng/mL IL-1β (Novoprotein, Shanghai, China) to induce chondrocyte degradation. Samples were collected at different time points (1, 2, 3, 6, 12, and 24 h), and the expression levels of ECM-related genes were analyzed by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), including aggrecan, type II collagen, ADAMTS-4, ADAMTS-5, and MMP-9.
3
Osteogenic Differentiation of PDLSCs
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H‐PDLSCs were seeded into six‐well plates at a density of 5 × 104 cells/well. When cells reached 80% confluence, IL‐1β (5 ng/mL; Novoprotein, Shanghai, China) and TNF‐α (10 ng/mL; Novoprotein) were added into normal α‐MEM medium or osteogenic differentiation induction α‐MEM medium. When cells reached 80% confluence in the six‐well plates, a nicotine sulphate solution (10−9 mol/L; Sigma‐Aldrich) and/or α‐BTX (10−8 mol/L; Tocris) was added to the culture medium. Cells were used for further experiments after indicated treatment periods.
4
Chondrocyte Cell Line C28/I2 Stimulation
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The human chondrocyte cell line C28/I2 (C28) was cultured in DMEM/F12 supplemented with 10% FBS (NEWZERUM, New Zealand), 100 U/ml penicillin, and 100 μg/mL streptomycin (MCE) in a humidified incubator at 37 °C in the presence of 5% CO2. Before treatment, chondrocytes were serum-starved for 12 h and then stimulated with recombinant human interleukin 1β (IL-1β, Novoprotein, China) at a concentration of 10 ng/mL for 48 h. The unstimulated chondrocytes were used as controls synchronously. After stimulation, the cells were washed with sterile PBS and collected.
5
Meningitic E. coli Infection of hBMECs
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Meningitic E. coli strain infection of primary hBMECs, as well as U251 cells and HUVECs, was performed in accordance with previously described methods61 62 (link). Briefly, E. coli overnight culture was resuspended and diluted in serum-free medium and added to the starved confluent primary hBMEC monolayer grown in 10-cm dishes at a multiplicity of infection of 10 (approximately 108 colony-forming units per dish) to allow invasion at 37 °C for 3 h. For cytokine stimulation, recombinant human IL-8, MIP-2, GRO-α, IL-1β, IL-6 and TNF-α were purchased from Novoprotein Scientific (Shanghai, China) and used at a final concentration of 10 ng/mL to stimulate the primary hBMECs for 24 h. Finally, cells were washed three times with chilled PBS and subjected to RNA extraction by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions.
6
Protocol for Dendritic Cell Induction
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Approximately 40-60ml of white cells was obtained from each patient using a blood-cell separator (FreseniusKabi, Germany). The yield of peripheral blood mononuclear cells (PBMCs) ranged from 1×109-3×109 cells. Cells were washed twice with AIM-V cell-culture medium (Gibco, America), transferred into culture flasks, and cultured for 2 hours. Adherent cells were used for induction of DCs by adding 100ng/ml GM-CSF (PeproTech, America) and 100ng/ml IL-4 (PeproTech, America) to the AIM-V cell-culture solution. Remaining non-adherent cells from the remainder of the PBMCs were stored at -80℃ and thawed for co-cultivation. On day 6, recombinant adenoviruses encoding different TAAs were used to infect DCs (MOI=10). On day 7, IL-1β (25 ng/ml; Novoprotein, China) and TNF-α (100 ng/ml; Novoprotein, China) were added into the culture for maturation of DCs.
7
Isolation and In-Vitro IVDD Model of Human Nucleus Pulposus Cells
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Human NP cells (HNPCs) were isolated from the normal IVD tissues. In short, the tissues were cut into sections of about 1 mm3 and treated for 0.5 hours with 0.25% trypsin (Gibco, Life Technologies, Paisley, UK), and then digested for 3 hours with 0.2% type II collagenase (Invitrogen, Carlsbad, CA, USA) at 37° C. After filtration and washing with PBS, the suspension was centrifuged, and the cells were cultured in the Dulbecco modified Eagle medium comprising F12 nutrient mixtures (Gibco, Grand Island, NY, USA), 15% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, St.Louis, MO, USA) at 37° C with 5% CO2. After fusion, HNPCs were digested and sub-cultured. The second-generation cells were applied for subsequent experiments.
An in-vitro model of IVDD was constructed by treating HNPCs with 10 ng/mL IL-1β (Novoprotein, Shanghai, China), 40 ng/mL TNF-α (PeproTech, East Windsor, NJ, USA), 500 μM hydrogen peroxide (H2O2) (349887, Sigma-Aldrich, MO) for 24 hours, respectively. Then, the cells were harvested for later experiments.
An in-vitro model of IVDD was constructed by treating HNPCs with 10 ng/mL IL-1β (Novoprotein, Shanghai, China), 40 ng/mL TNF-α (PeproTech, East Windsor, NJ, USA), 500 μM hydrogen peroxide (H2O2) (349887, Sigma-Aldrich, MO) for 24 hours, respectively. Then, the cells were harvested for later experiments.
8
Bilobalide Effects on Cell Viability
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Bilobalide (purity ≥ 98%) was purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Requirements considered to be relevant in guidelines for best practice in natural products pharmacological research have been taken into account (Heinrich et al., 2020 (link)). Viable cell proliferation was determined using CCK-8 (APExBIO, Houston, TX, United States). Cells were seeded in 96-well plates and treated with 10 ng/ml IL-1β (Novoprotein, China) and Bilobalide (0, 7.5, 15, 30, 60, and 120 μM) for 12 or 24 h. Different concentrations of Bilobalide dissolved in dimethylsulfoxide (DMSO) were added to the medium, with the final concentration of DMSO not exceeding 0.05%. Following that, 10 μl of CCK-8 solution was added. After 30 min, a multi-function microplate reader (BioTEK, Winooski, VT, United States) was used to measure the absorbance at 450 nm.
9
Isolation and Culture of Chondrocytes and Bone Marrow Stem Cells
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Mouse chondrocyte (ADTC5) was purchased from KeyGEN (Jiangsu, China) and grown in a 1:1 mixture of DMEM and Ham's F12 (Gibco, Massachusetts, United States) containing 5% fetal bovine serum (Gibco, Massachusetts, United States), 2 mM Glutamine (Gibco, Massachusetts, United States), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Massachusetts, United States) at 37 °C in an atmosphere with 5% CO2. ADTC5 cells were stimulated with IL1β (10 ng/ml) (Novoprotein, Suzhou, China) for 24 hours to mimic OA in vitro and then harvested for further studies. 12 Sprague Dawley (SD) rats (60-80 g) were purchased from the Laboratory Animal Center of Guangzhou University of Chinese Medicine (License number: SCXK 2018-0047, quality certificate number: 44005900002750). Animals were euthanized by injection of sodium pentobarbital (200 mg/kg body weight). Tibia and femur were isolated under sterile conditions, and the bone marrow was flushed out with DMEM basic containing 1% double antibody. Cells were collected through centrifugation at 1000 rpm for 10 minutes at room temperature. Bone marrow mesenchymal stem cells (BMSCs) were cultured with SD rat BMSCs basal medium (Cyagen, Shanghai, China) in an atmosphere of 5% CO2 at 37 °C. P3 and P4 generations of BMSCs can be used for subsequent experiments.
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