The fresh PRP samples were incubated without or with either oxLDL or native LDL (oxLDL, 2 mg/mL, cat# BT-910; nLDL, 5 mg/mL, cat# BT-903, Biomedical Technologies Inc., MA, USA) at increasing concentrations: 0-, 10-, 20-, and 40 μg/mL for 30 min at room temperature (RT). The oxLDL was generated by copper sulfate oxidation of LDL. The lot used in this study migrated 2.1-fold further than LDL on an agarose gel, and it was used for experiments within 4 weeks. The lowest concentration of 10 μg/mL corresponds to physiological levels found in individuals without metabolic syndrome (13 (link)). Plasma samples were further centrifuged, as described in Figure 1 , and stored at −80°C until MV analysis.
Ox ldl
Manufactured by Biomedical Technologies
Sourced in United States
Ox-LDL is a laboratory equipment used for the detection and measurement of oxidized low-density lipoprotein (LDL) in biological samples. It is a tool utilized in research and clinical settings to study the role of oxidized LDL in various health conditions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Faststart universal sybr green master rox, by Roche (1 mentions)
Acetylated ldl ac ldl, by Biomedical Technologies (1 mentions)
Simettl3 1, by GenePharma (1 mentions)
Simettl3 2, by GenePharma (1 mentions)
Oil red o, by Merck Group (1 mentions)
Si nc, by GenePharma (1 mentions)
Huvecs, by Lonza (1 mentions)
Hcaec, by Lonza (1 mentions)
Skimmed milk powder, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Pyrobest dna polymerase, by Takara Bio (1 mentions)
Rt pcr kit, by Qiagen (1 mentions)
Stigmasterol, by Merck Group (1 mentions)
Rnase inhibitor, by Merck Group (1 mentions)
16 protocols using ox ldl
1
Incubation of PRP with oxLDL and nLDL
2
Macrophage response to oxLDL
3
Fetal RPE Response to Lipids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human fetal RPE or ARPE-19 cells, grown at postconfluence for 2 to 4 weeks, were serum-starved overnight and treated for 24, 48, and 72 hours with LDL or ox-LDL (Biomedical Technologies, Alfa Asear, LLC, Ward Hill, MA, USA) suspended in serum-free media. Conditioned media and cell lysates were collected.
4
Lipoproteins and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human lipoproteins [Ox-LDL, acetylated-LDL (Ac-LDL), indocarbocyanine dye (Dil)-labeled Ox-LDL, and HDL] were obtained from Biomedical Technologies Inc. (Stoughton, MA). Transcriptor First Strand cDNA Synthesis kit and FastStart Universal SYBR Green Master (Rox) were obtained from Roche (Basel, Switzerland). Chemicals were obtained from Sigma-Aldrich (Saint Louis, MO) unless stated otherwise.
5
Regulation of Endothelial Cell Function by IGF2BP1 and METTL3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ox-LDL was obtained from Biomedical Technologies, Inc. (Stoughton, MA, United States). Oil red O was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, United States). Small interference RNAs (siRNA) targeting IGF2BP1 (si-IGF2BP1#1, si-IGF2BP1#2, and si-IGF2BP1#3), METTL3 (si-METTL3#1, si-METTL3#2, and si-METTL3#3), and a scrambled siRNA (si-NC) were obtained from GenePharma Co., Ltd. (Shanghai, China). The siRNA sequences were shown in Table 1 . The recombinant plasmid expressing JAK2 (pcDNA-JAK2) or IGF2BP1 (pcDNA-IGF2BP1) was synthesized by GeneCreate Biological Engineering Co., Ltd. Plasmids or siRNAs were transfected into HUVECs using jetPRIME transfection reagent (Poluplus-transfection Inc., New York, NY, United States) according to the instructions of the manufacturer. For double transfection, DNA (2 μg) and siRNA (final concentration: 50 nM) were mixed and co-incubated with 5 μl jetPRIME reagent for 15 min at room temperature in 6-well plates. Next, the transfection mix was added to cells in serum-containing medium dropwise. At 24 h after transfection, the transfection medium was replaced with the refresh cell growth medium.
6
THP-1 Monocyte Adhesion to Activated HUVEC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
THP-1 monocytes were washed in PBS and were then labeled for 15 min with CFDA-SE (Molecular Probes for Life Technologies, Carlsbad, CA, USA). At the end, cells were re-suspended in fresh medium and were incubated for 30 min at 37 °C. Labeled THP-1 were added (for 1 h, at 37 °C) to unactivated or oxidized LDL-activated (ox-LDL 100 μg/ml, 16 h, Biomedical Technologies Inc.) HUVEC monolayers. THP-1 adhesion was visualized using an inverted microscope.
7
Endothelial Cell Cultures for Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), and human coronary artery endothelial cells (HCAECs) were purchased from Lonza (Basel, Switzerland) and cultured as described previously.34 (link) Population-doubling levels (PDLs) were calculated as described previously;35 (link) briefly, the number of population doublings (PD) that occurred between passages was calculated according to the equation PD=log2(Ch/Cs), where Ch is the number of viable cells at harvest and Cs is the number of cells seeded. All experiments were performed at the PDLs indicated in the text. Cells were incubated at 37 °C, 16 h, in the presence of 100 μg/ml ox-LDL (Biomedical Technologies Inc., Stoughton, MA, USA).
8
Western Blot Analysis of Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis buffer, polyvinylidene difluoride (PVDF) membranes, Tween20, hematoxylin, paraformaldehyde, RNase inhibitor, skimmed milk powder, Oil red O solution, BCA kit, trichloroacetic acid, stigmasterol, isopropanol, acetonitrile, IFN-γ, and lipopolysaccharide (LPS) were purchased from Sigma (St. Louis, MO, USA). OxLDL was purchased from Biomedical Technologies (Stoughton, MA, USA). DNase, the RT-PCR kit, and the Qiagen RNeasy kit were purchased from Qiagen (Hilden, Germany). Rabbit antihuman ATF3, TN-C, IL-6, IL-8, TLR-4 antibodies, anti-β-actin antibody, and goat anti-rabbit IgG-horseradish peroxidase (HRP) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Pyrobest DNA polymerase and avian myeloblastosis virus (AMV) reverse transcriptase were purchased from Takara Biotechnology (Dalian, China). Furthermore, fetal bovine serum (FBS), Lipofectamine TM 2000, L-glutamine, penicillin, streptomycin, RPMI 1640 medium, and TRIzol were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). BCA Protein Assay Kits and Enhanced Chemiluminescence (ECL) TM Western Blotting Detection Reagents were purchased from Thermo Pierce (Rockford, IL, USA). The SYBR® Green PCR Master Mix was purchased from Applied Biosystems (Foster City, CA, USA).
9
Modulating miR-144-5p in Atheroprone HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVECs were acquired from the Shanghai Institute of Life Sciences, Chinese Academy of Sciences. Culture media for HUVECs contained routine medium 199 (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (unless otherwise stated) (Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.), 1% endothelial cell growth supplement (Sigma-Aldrich; Merck KGaA) and 10 ng/ml epidermal growth factor in a 5% CO2 humidified atmosphere at 37°C. To mimic atheroprone conditions, HUVECs were exposed to proatherogenic oxLDL (25 µg/ml; Biomedical Technologies S.L.).
HUVECs (5.0×104 cells/well) were transfected with 100 nM miR-144-5p mimic or 100 nM mimic control for 48 h at 37°C using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 h of transfection, the transfection efficiency was detected using reverse transcriptase quantitative PCR (RT-qPCR).
HUVECs (5.0×104 cells/well) were transfected with 100 nM miR-144-5p mimic or 100 nM mimic control for 48 h at 37°C using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 h of transfection, the transfection efficiency was detected using reverse transcriptase quantitative PCR (RT-qPCR).
10
Oxidized LDL Uptake in RPE Cells
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!