Bone marrow-derived DCs (BMDCs) were obtained from C57 BL/6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). After the mice were euthanized using CO2 inhalation, the femurs of both pelvic limbs were removed, and the bone marrow cavity was flushed to obtain bone marrow cells. Bone marrow progenitor cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF) (R & D Systems, Minneapolis, MN, USA) and 1 ng/ml IL-4 (R & D Systems, Minneapolis, MN, USA). The non-adherent cells were gently washed out after 24 h, and the remaining loosely adherent clusters were cultured. The culture medium was changed every other day. On day six, the cells were stimulated using 50ug/ml oxidized low-density lipoprotein (oxLDL) (Serotec, UK) for 48 h, with or without 200 ug/ml AGEs-BSA (BioVision, Palo Alto, CA, USA) interference to simulate the high-lipid and high-glucose environment of ApoE−/− mice, respectively.
Ages bsa
Manufactured by Abcam
Sourced in United States
AGEs-BSA is a lab product that consists of bovine serum albumin (BSA) modified with advanced glycation end products (AGEs). It can be used as a model or reference material in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (2 mentions)
C57bl 6 mice, by Charles River Laboratories (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Granulocyte macrophage colony stimulating factor gm csf, by R&D Systems (1 mentions)
Cck 8 solution, by Dojindo Laboratories (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Vi cell xr, by Beckman Coulter (1 mentions)
Quercetin, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Norepinephrine, by Merck Group (1 mentions)
Claycomb medium, by Merck Group (1 mentions)
Fibronectin, by Corning (1 mentions)
Plasmid plus midi kit, by Qiagen (1 mentions)
Protein g bead, by Roche (1 mentions)
Ezviewtm red anti ha affinity gel, by Merck Group (1 mentions)
Hmgb1, by Abcam (1 mentions)
Ha tag c29f4, by Cell Signaling Technology (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Alpelisib, by MedChemExpress (1 mentions)
Trametinib, by MedChemExpress (1 mentions)
8 protocols using ages bsa
1
BMDC Differentiation and Stimulation
2
Evaluating the Effects of TNF-α and AGEs on PDLSCs
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Refer to the previous experimental results and the purpose of the experimental group (the concentration of TNF-α cannot cause a large amount of cell apoptosis), commercially available TNF-α (Pepro Tech, USA) at a concentration of 10 UG/mL was diluted to an experimental concentration of 10 ng/mL with PBS containing 1% BSA, and the experimental concentration of AGEs-BSA (Bio-Vision, USA) was 100 μg/mL. The density of the third generation PDLSCs was adjusted to 2.0×104 cells/mL and inoculated into 96-well plates, four plates per plate, five wells per group. After twenty-four hours, the medium was changed to normal α-MEM (Control group), α-MEM containing 10 ng/mL TNF-α (TNF-α-PDLSCs group), α-MEM containing 100 μg/mL AGEs-BSA (AGEs-PDLSCs group), α-MEM containing 100 μg/mL AGEs-BSA and 10 ng/mL TNF-α (AGEs+TNF-α-PDLSCs group), and cultured at 37 °C in a 5% CO2 incubator. After twenty-four hours, a plate was randomly selected and incubated with CCK-8 solution (DOJINDO, Japan) for one hour. The absorbance was measured at 450 nm with a microplate reader (A5082-TECAN, USA) for seven days. Count the number of cells using a cell analyzer (Beckman Vi-Cell XR, USA). Calculate cell proliferation curve based on data.
3
Gingival Fibroblast Cultures and AGEs
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All steps were carried out in compliance with the authorized guidelines from the Institutional Review Board at the Chung Shan Medical University Hospital. HGFs from two healthy individuals were obtained after crown lengthening procedure using an explant technique as previously mentioned.31 (link) In this investigation, cell cultures of two individual HGFs between the third and eighth passages were employed. Advanced Glycation End-Products (AGEs)-BSA was purchased from BioVision (Milpitas, CA, USA) and Quercetin was obtained from Sigma Chemical Co. (St. Louis, MO, USA).
4
HL-1 Cell Culture and AGEs-BSA Treatment
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HL‐1 cells were obtained from the laboratory of Dr. William Claycomb (Louisiana State University Health Science Center, New Orleans, LA). Cells were cultured in Claycomb medium (51,800C; Sigma‐Aldrich), supplemented with 10% fetal bovine serum (FBS) (F8687; Sigma), 2 mmol/L L‐glutamine (25,030,081; Gibco), 100 μmol/L norepinephrine (HY‐137,158; Sigma) on flasks precoated with 5 μg/ml fibronectin (356,008; Corning) and 0.02% gelatin (G‐9382; Sigma), then incubate at 37°C in a humidified atmosphere of 5% CO2.
AGEs‐BSA were purchased from Abcam (ab51,995, UK), which was prepared by reacting bovine serum albumin with glycolaldehyde under sterile conditions as described previously. Cells were treated with drugs when the cell fusion reached about 60%–70%. HL‐1 cells were incubated with Claycomb medium plus 10% FBS in the presence of 2 μg/ml anti‐RAGE antibody for 3 h, and then 400ug/ml AGEs‐BSA was added and cultured for 48 h, BSA (2221‐BSA, BioVision) served as the negative control group.
AGEs‐BSA were purchased from Abcam (ab51,995, UK), which was prepared by reacting bovine serum albumin with glycolaldehyde under sterile conditions as described previously. Cells were treated with drugs when the cell fusion reached about 60%–70%. HL‐1 cells were incubated with Claycomb medium plus 10% FBS in the presence of 2 μg/ml anti‐RAGE antibody for 3 h, and then 400ug/ml AGEs‐BSA was added and cultured for 48 h, BSA (2221‐BSA, BioVision) served as the negative control group.
5
Biochemical Reagents and Antibodies Used
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AGEs-BSA (#2221-10) was purchased from Biovision (Milpitas, CA, USA). The recombinant mouse HMGB1 was purchased from Abcam (#ab181949). Antibodies against ERK1/2 (#9102), phospho-ERK1/2 (Thr202/Tyr204) (#9101), SAPK/JNK (#9252), phospho-SAPK/JNK (Thr183/Tyr185) (#9251), p38 MAPK (#9212), and phospho-p38 MAPK (Thr180/Tyr182) (#9211) and antibodies against phospho-IKKα/β (Ser176/180) (#2679), SLP76 (#4958), and the HA tag (C29F4) (#3724) were purchased from Cell Signaling Technology (Boston, MA, USA). Polyclonal antibodies against RAGE (N-16) (#sc-8230) and IKKα/β (H-470) (#sc-7607) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-Flag tag (#F3165) monoclonal antibodies, ANTI-FLAG® M2 affinity gel (#A2220) and EZviewTM Red anti-HA affinity gel (#E6779) were obtained from Sigma (St. Louis, MO, USA). siRNAs targeting SLP76 (#sc-36502) and RAGE (#sc-36375) and the corresponding control siRNAs (#sc-142627) were obtained from Santa Cruz Biotechnology. Protein G beads (#11719416001) were obtained from Roche (Mannheim, Germany), and a Plasmid Plus Midi Kit (#12945) for DNA preparation was purchased from Qiagen (Hilden, Germany). TAT and TAT-SAM peptides were synthesized by Bootech BioScience & Technology (Shanghai, China).
6
AGEs-BSA Compound Preparation Protocol
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We purchased AGEs-BSA (AGEs) from Abcam (DBA, Milan, Italy); the FPS-ZM1, BSA and N-acetyl-L-cysteine (NAC) from Merck Life Science (Milan, Italy). The Trametinib, Alpelisib and Reparixin were obtained from MedChemExpress (DBA, Milan, Italy). The anti-IL-8 neutralizing antibody (MAB208) was acquired from R&D Systems (Bio-Techne, Milan, Italy). All of the compounds were solubilized in dimethyl sulfoxide (DMSO), except for AGEs-BSA and BSA that were dissolved in phosphate-buffered saline (PBS), and NAC that was solubilized in water.
7
Investigating AGE-Induced Macrophage Polarization
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Melatonin, fetal bovine serum (FBS) and trypsin-EDTA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and penicillin–streptomycin were purchased from Gibco Life Technologies (Grand Island, NY, USA). The antibodies of advanced glycosylation end products (AGEs) of bovine serum albumin (AGEs-BSA), primary antibodies against CD68 (ab283654), CD86 (ab239075), nitric oxide synthase 2 (iNOS2, ab283655) and ARG (arginase Type II, ab264066) were purchased from Abcam (Cambridge, UK). PE anti-mouse CD86 (159204) and FITC anti-mouse CD206 (141703) were purchased from BioLegend (San Diego, CA, USA), and alpha-smooth muscle actin (α-SMA) (BM0002) was purchased from Boster Biological Technology (Wuhan, China). Masson’s Staining Kit (G1340) was purchased from Solarbio (Beijing, China).
8
HK-2 Cell Culture with AGEs-BSA and EMPA
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This work obtained HK-2 cells in American Type Culture Collection (Manassas, VA, USA), then cultivated them following the method described elsewhere [11 (link)]. All assays were conducted within serum-free RPMI 1640 medium containing 0.2% bovine serum albumin (BSA, fatty acid free; SigmaAldrich; Merck KGaA). We obtained EMPA in MedChemExpress (Shanghai, China) and AGEs-BSA from Abcam (Cambridge, UK). HK-2 cells were cultivated in experimental medium including 200 µg/mL AGEs-BSA, 500 nM EMPA, or the combination of these two for 48-h.
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