K1 or BCPAP cells stably overexpressing METTL16 or with METTL16 knocked down and their corresponding control cells were crosslinked by UV (260 nm, 130 mJ/cm2) and harvested in cold PBS. RNA immunoprecipitation (RIP) was performed using a Magna RIP Kit (17–700, Millipore). Briefly, harvested cells were lysed (10% for input) and incubated with an anti-YTHDC2 antibody (1:1000; ab220160; Abcam) overnight at 4 °C. After washing, the immunoprecipitated complex was digested with proteinase K. RNA was extracted, detected via qRT‒PCR and normalized to the input. For m6A RNA binding experiments, total RNA from K1 cells stably overexpressing METTL16 or with METTL16 knocked down was treated with deoxyribonuclease I (Solarbio, China). The RNAs were sonicated and precipitated with Protein G Magnetic Beads (S1430S, NEB) bound to a m6A antibody (202,003, SYSY). After proteinase K (10 µg/mL) enzymolysis, RNAs were isolated for qRT‒PCR analysis (the input served as a control). For m6A sequencing, m6A-RNAs were acquired from the aforementioned m6A-RIP assay. The RNA libraries were created via quality inspection and subsequently subjected to analysis on an Illumina HiSeq instrument. The peaks were visualized with IGV software.
Deoxyribonuclease 1
Manufactured by Solarbio
Sourced in China
Deoxyribonuclease I is an enzyme that catalyzes the hydrolytic cleavage of DNA. It is a type of endonuclease that cleaves the phosphodiester bonds in the DNA backbone, resulting in the formation of shorter DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Type 2 collagenase, by Solarbio (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Dmem lg, by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Ab220160, by Abcam (1 mentions)
Magna rip kit, by Merck Group (1 mentions)
Protein g magnetic beads, by New England Biolabs (1 mentions)
Hiseq instrument, by Illumina (1 mentions)
Edta 2na, by Sangon (1 mentions)
Forma 3110, by Thermo Fisher Scientific (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Trypsin, by Sangon (1 mentions)
70 μm filter, by BD (1 mentions)
Cd8a clone 53 6, by BD (1 mentions)
Collagenase 1, by Solarbio (1 mentions)
Ifnγ clone xmg1, by BioLegend (1 mentions)
Pd 1 clone rmp1 30, by BioLegend (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Collagenase 3, by Worthington (1 mentions)
7 protocols using deoxyribonuclease 1
1
METTL16-mediated m6A RNA Immunoprecipitation
2
Isolation and Culture of hAMSCs and BMSCs
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Following a previously described method (Wang et al., 2020 (link)), hAMSCs were isolated from the placental amnion of healthy pregnant women, using collagenase type II (Solarbio) and deoxyribonuclease I (Solarbio) after obtaining informed consent. According to the whole bone‐marrow culture methods described by Yang et al. (2017 (link)), BMSCs were isolated from mice treated as described in Section 4.3 . The hAMSCs and BMSCs were maintained at 37°C in a 5% (v/v) CO2 incubator (Forma 3110; Thermo) with a humidified atmosphere. Their culture medium consisted of low‐glucose Dulbecco's modified Eagle's medium (LG‐DMEM; Gibco), 10% fetal bovine serum (FBS; Gibco), 1% non‐essential amino acids (Gibco), 1% l ‐alanyl‐glutamine dipeptide (l ‐GlutaMAX) (Gibco), and 10 ng/ml basic fibroblast growth factor (Peprotech). The culture medium was refreshed every 3 days. The growth of the cells was observed under an inverted microscope (Olympus). When the cells reached about approximately 80% confluency, they were passaged using 0.125% trypsin (including 0.02% EDTA‐2Na) (Sangon Biotech). BMSCs and hAMSCs at passages 2 (P2) and 3 (P3), respectively, were used for further experiments.
3
Neuroprotective Effects of Edaravone Dexborneol and DNase I in MCAO Mice
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Animals were randomly assigned to four groups: Sham-operated and vehicle-treated group (Sham group), distal middle cerebral artery occlusion+vehicle-treated group (MCAO group), distal middle cerebral artery occlusion+Edaravone Dexborneol-treated group (Eda.B group), and distal middle cerebral artery occlusion+DNase I-treated group (DNase I group).
Edaravone Dexborneol was provided by Simcere Pharmaceutical Group Co. Ltd. Mice in the Eda.B group received intravenous (i.v.) Eda.B at 7.5 mg/kg instantly and every 12 h after MCAO. The dose of medicine was based on a previous study [14 (link)]. Mice in the DNase I group were injected intravenous (i.v.) Deoxyribonuclease I (Solarbio, China) at 10 mg/kg instantly and every 12 h after MCAO. Mice in the Sham group and MCAO group were injected with the same amount of normal saline at the same time. At days 1 and 3 after MCAO, according to the experimental plan, animals were killed quickly by decapitation under deep anesthesia, and samples were collected for follow-up studies.
Edaravone Dexborneol was provided by Simcere Pharmaceutical Group Co. Ltd. Mice in the Eda.B group received intravenous (i.v.) Eda.B at 7.5 mg/kg instantly and every 12 h after MCAO. The dose of medicine was based on a previous study [14 (link)]. Mice in the DNase I group were injected intravenous (i.v.) Deoxyribonuclease I (Solarbio, China) at 10 mg/kg instantly and every 12 h after MCAO. Mice in the Sham group and MCAO group were injected with the same amount of normal saline at the same time. At days 1 and 3 after MCAO, according to the experimental plan, animals were killed quickly by decapitation under deep anesthesia, and samples were collected for follow-up studies.
4
Isolation and Characterization of hAMSCs
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As per previously described methods [26 (link), 27 (link)], hAMSCs were isolated from placental amnion tissue that was collected from normal pregnant women after gaining informed consent using collagenase type II (Solarbio, Beijing, China) and deoxyribonuclease I (Solarbio, Beijing, China) enzymes. The hAMSCs were cultured in Dulbecco's modified Eagle medium low glucose (LG-DMEM) (Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, New York, USA), 1% nonessential amino acids (Gibco, New York, USA), 10 ng/mL basic fibroblast growth factor (bFGF) (Peprotech, NJ, USA), and 1% L-alanyl-L-glutamine dipeptide (L-GlutaMAX) (Gibco, New York, USA) in a humid atmosphere of 5% CO2 at 37°C. Culture medium was replaced by fresh medium every three days. When the cells reached 80% confluency, the harvested cells were passaged. Cells that belonged to passage 2 (P2) were used for further analysis in the study. Subsequently, these hAMSCs were analyzed using flow cytometry and the immunocytochemical staining method according to the protocols described in previous studies [26 (link), 27 (link)]. This research was conducted in accordance with the Declaration of Helsinki and the guidelines of the Ethical Committee of the Affiliated Hospital of Zunyi Medical University (Zunyi China).
5
Tumor-Infiltrating Immune Cell Analysis
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Tumors were removed from sacrificed mice and digested by collagenase I (1.5 mg/ml) and deoxyribonuclease I (100 μg/ml; Solarbio) in RPMI 1640 for 1 hour at 37°C. The cell suspensions were passed through 70-μm filters (Falcon) to remove undigested tumor tissues, and then, the erythrocytes were removed by ACK lysis buffer. Cell suspensions were incubated in mouse Fc block (anti-CD16/CD32, BioLegend) before staining. Fluorochrome-conjugated anti-mouse CD45.2 (clone 104, eBioscience), CD8a (clone 53-6.7, BD Biosciences), IFNγ (clone XMG1.2, BioLegend), and PD-1 (clone RMP1-30, BioLegend) antibodies were used following the manufacturer’s protocol. Flow cytometry results were analyzed using FlowJo software.
6
Lung Single-Cell Suspension Preparation
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Washed right lung tissues were dissociated into single-cell suspensions as described previously, with certain modifications (33 (link)-35 (link)). Lung tissues were minced with sterilized scissors and forceps [RNase was inactivated by high temperature (121°C)], and were enzymatically digested with RPMI-1640 medium (cat. no. 11875085; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 1% BSA (Beijing Solarbio Science & Technology Co., Ltd.), 10 mmol/l HEPES, 60 units/ml deoxyribonuclease I (cat. no. D4527), 1 mg/ml type XI collagenase (cat. no. C7657), 1,000 U hyaluronidase (cat. no. H3631), and 0.1 mg/ml Kunitz-type soybean trypsin inhibitor (cat. no. 7659; Sigma-Aldrich; Merck KGaA), and the tissues were agitated for 40 min in a 37°C incubator. Following digestion, the lung tissue and supernatant were filtered through a 200-gauge stainless steel mesh to remove cell clumps and undigested tissue. The filtered suspension was centrifuged at 200 × g for 10 min at room temperature, and lung cells were resuspended in FACS buffer (cat. no. 00-4222-57; Thermo Fisher Scientific, Inc.) at a concentration of ~2×106/ml. The number of live cells was measured using light microscopy at low resolution (magnification, ×40) by staining cells with 0.4% Trypan Blue for 10 min at 22-25°C.
7
Endometrial Cell Isolation and Culture
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Endometrial single-cell isolation was carried out as described [9 (link)]. Endometrial tissues were collected and the following procedure was performed with 24 h of the collection. Tissues were minced into pieces smaller than 1 mm and suspended in PBS containing collagenase III (0.3 mg/mL, Worthington, Lakewood, NJ, USA) and deoxyribonuclease I (40 ug/mL, SolarBio, Beijing, China). Tissue suspension was digested at 37 °C in water bath under constant shaking. Red blood cells were removed by density-gradient centrifugation using Ficoll-Paque (GE Healthcare, Berlin, Germany). Leukocytes were excluded using anti-CD45 microbeads (Miltenyi Biotec Inc., Bergisch Gladbach, Germany). Negative cells were subjected to subsequent isolation for epithelial and stromal cells. Epithelial cells were selected using anti-CD326 microbeads (Miltenyi). Negative cells were considered as stromal cells. Stromal cells were cultured in fibronectin-coated plates (1 mg/mL, Invitrogen). Culture media contained DMEM/F12, 10% FBS, 1% antibiotics, and 2 mmol/L L-glutamine (Invitrogen, Carlsbad, CA, USA). Cell culture was incubated in a humidified carbon dioxide (5%) incubator at 37 °C.
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