BeNa Culture Collection supplied the hRMECs (BNCC, Beijing) and cultured them in an endothelial cell medium (ECM) containing 10% fetal bovine serum (FBS) with 5% CO2 at 37°C. Glucose 30 mM cell stimulation (high-glucose, HG). According to the manufacturer’s recommendations, hRMECs were separated into groups for transfection with Lipofectamine 3000 (Invitrogen).
Hrmecs
Manufactured by Thermo Fisher Scientific
Sourced in United States
HRMECs are human renal microvascular endothelial cells. They are primary cells derived from human renal tissue and are used for in vitro studies of renal microvascular endothelial function.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Hrmecs, by Angio-Proteomie (2 mentions)
Hrmecs, by Procell (1 mentions)
Hrmecs, by Merck Group (1 mentions)
Lipofectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
Endothelial cell culture medium, by ScienCell (1 mentions)
Hyperfect, by Qiagen (1 mentions)
Silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Modular incubator chamber, by Thermo Fisher Scientific (1 mentions)
Microvascular endothelial cell growth kit bbe, by American Type Culture Collection (1 mentions)
Complete human endothelial cell medium, by Cell Biologics (1 mentions)
Hrmecs, by Cell Biologics (1 mentions)
Newborn calf serum ncs, by Thermo Fisher Scientific (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Lentivirus, by Geneseed (1 mentions)
Mir 20b 5p inhibitor, by RiboBio (1 mentions)
Mir nc mimic, by RiboBio (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by ScienCell (1 mentions)
6 protocols using hrmecs
1
High-Glucose Stimulation of hRMECs
2
High Glucose-induced HMGB1 Regulation in HRMECs
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HRMECs were purchased from Procell Life Science&Technology (Wuhan, China). The HRMECs were grown on the poly‐L‐lysine‐coated plates in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, endothelial cell growth factor, heparin, and hydrocortisone in a 5% CO2‐enriched atmosphere with constant humidity. To maintain uniform conditions, all experiments were performed using passage 5 HRMECs. HRMECs were grown in the 50 mM glucose medium (Sigma‐Aldrich) for 48 h. D‐Mannitol was used as an osmotic control. For the treatment, NK (1 μM and 2 μM) was pretreated for 2 h before HG stimulation. For HMGB1 silencing, siRNA constructs were diluted in buffer (Lipofectamine transfection reagent; Invitrogen Inc., Carlsbad, CA, USA) and transfected in culture medium of HRMECs at a working concentration of 100 nM for 24 h before NK treatment. Scrambled siRNA was used as control. The sequences for HMGB1‐siRNA were 5′‐GGC UUU CAC UUA AGA ACU UTT‐3′.
3
Investigating GPX4 Knockdown in Human Retinal Endothelial Cells
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Human retinal microvascular endothelial cells (HRMECs; Cell Systems, WA, USA) were cultured in an endothelial cell culture medium (ScienCell, CA, USA) containing 30 mM D-glucose, 10% fetal bovine serum (FBS; ScienCell), and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin). The cells were cultured in a 5% CO2 incubator at 37 °C.
On reaching 60–70% confluency, HRMECs were transfected with GPX4 siRNA (Silencer select siRNA, ID# s6112; Thermo Scientific, MA, USA) or the negative control, Silencer select negative control #1, in combination with Hyperfect (Qiagen, CA, USA). After 24 h, the medium was changed and Knockdown efficiency was confirmed by Western blotting. Then cells were treated with porous Se@SiO2 nanospheres or NPs after maintaining the cells for an additional 24 h. After three days of incubation with a high-glucose (HG; 30 mM d-glucose) medium, further experiments were conducted as described below.
On reaching 60–70% confluency, HRMECs were transfected with GPX4 siRNA (Silencer select siRNA, ID# s6112; Thermo Scientific, MA, USA) or the negative control, Silencer select negative control #1, in combination with Hyperfect (Qiagen, CA, USA). After 24 h, the medium was changed and Knockdown efficiency was confirmed by Western blotting. Then cells were treated with porous Se@SiO2 nanospheres or NPs after maintaining the cells for an additional 24 h. After three days of incubation with a high-glucose (HG; 30 mM d-glucose) medium, further experiments were conducted as described below.
4
Culturing Human Retinal Endothelial Cells
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Human primary retinal microvascular ECs (HRMECs) were obtained from Cell Biologics (Cat. No. H-6065; Chicago, IL, USA) and used between passages 3-8. The type of cells and no pathogen (including mycoplasma) contamination were confirmed by the supplier.
HRMECs were cultured in Vessel Cell Basal Medium (VCBM, ATCC, Manassas, VA, USA) supplemented with Microvascular Endothelial Cell Growth Kit-BBE (ATCC), and 1% penicillin/streptomycin, or in Complete Human Endothelial Cell Medium (Cell Biologics). In some experiments, HRMECs were incubated with 20 ng/ml recombinant hVEGF, 10 μM DAPT, 200 μM CoCl2, or 20-100 µM adenosine in the presence of 10 µM EHNA according to the protocol previously described58 (link), 59 (link). For the experiments requiring hypoxia, HRMECs were placed in a modular incubator chamber (Thermo Scientific, Waltham, MA) with 0.1–2% O2.
HRMECs were cultured in Vessel Cell Basal Medium (VCBM, ATCC, Manassas, VA, USA) supplemented with Microvascular Endothelial Cell Growth Kit-BBE (ATCC), and 1% penicillin/streptomycin, or in Complete Human Endothelial Cell Medium (Cell Biologics). In some experiments, HRMECs were incubated with 20 ng/ml recombinant hVEGF, 10 μM DAPT, 200 μM CoCl2, or 20-100 µM adenosine in the presence of 10 µM EHNA according to the protocol previously described58 (link), 59 (link). For the experiments requiring hypoxia, HRMECs were placed in a modular incubator chamber (Thermo Scientific, Waltham, MA) with 0.1–2% O2.
5
Culturing Endothelial and HEK293 Cells
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HRMECs were obtained from Angio-Proteomie (Peabody, MA, United States). HRMECs were cultured in endothelial cell medium (ECM) supplemented with 5% Newborn Calf Serum (NCS; Life Technologies), 20 μg/ml of ECGS, 100 μg/ml streptomycin, and 100 U/ml penicillin in a T-25 flask coated with 8.5 μg/ml of BPF at 37°C with 5% CO2. When the cells became 90% confluent, they were subcultured at a 1:3 ratio. HEK293 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in DMEM supplemented with 10% NCS and incubated at 37°C with 5% CO2.
6
Investigating miR-20b-5p and circDNMT3B in HRMECs
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HRMECs (Angio-Proteomie, Boston, Mass, USA) were cultured in endothelial cell medium (ECM) with 5% fetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin/streptomycin solution (ScienCell Research Laboratories, Carlsbad, CA, USA) at 37 °C under 5% CO2 atmosphere. Cells between passages three and eight were used in this study. Cells were treated with 5 mM glucose as a normal glucose (NG) control, 5 mM glucose plus 25 mM mannitol as an osmotic control, or 30 mM glucose as a high-glucose (HG) treatment. miR-20b-5p inhibitor, miR inhibitor negative control (NC), miR-20b-5p mimic, or miR mimic NC (RiboBio, Guangzhou, China) was transfected into HRMECs using Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Small interfering BMP and activin membrane-bound inhibitor (siBAMBI) and siRNA NC synthesized by RiboBio were transfected using Lipofectamine RNAiMAX (Life Technologies). The human circDNMT3B sequence was inserted into the pLCDH-ciR vector, and then incorporated into a lentivirus by Geneseed Biotech (Guangzhou, China). To induce stable circDNMT3B-overexpressing HRMECs, the HRMECs at passage three were infected with lentivirus containing circDNMT3B or ciR NC (empty vector). All sequences used in this study are listed in Supplementary Table S1.
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