JQ1 or vehicle was intravitreally injected to rd10 mice at PN14, as described above. After 10 days, retinal microglial cells were isolated and purified, following our published method with minor modifications [7 (link)]. Briefly, animals were euthanized and their eyeballs enucleated immediately. The globes were dissected free of periocular connective tissues and rinsed with HBSS buffer. The anterior segment and vitreous were removed, and the retina was dissected free from the underlying RPE layer. The retinas were transferred into DMEM containing 70 U/ml collagenase (0.5 ml per eye) and incubated at 37 °C for 60 min. Enzyme activity was terminated using DMEM containing 10% FBS. The retinas were dissociated mechanically and passed through 40-μm nylon mesh (Corning, NY). The dissociated cells were then labeled with antibodies for CD11b (BD, 557397) and CD45 (BD, 559864) and DAPI. Microglial cells were purified by flow sorting (CD11b positive and CD45 low) and a >95% purity was achieved. Cells were used immediately for RNA isolation and qRT-PCR.
40 μm nylon mesh
Manufactured by Corning
Sourced in United States
The 40-μm nylon mesh is a laboratory equipment product designed for filtration and separation applications. It features a mesh size of 40 micrometers, made from nylon material. This product serves as a physical barrier, allowing the passage of smaller particles while retaining larger ones, making it suitable for various filtration and separation processes in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Mannitol, by Merck Group (1 mentions)
L glucose, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
D glucose, by Merck Group (1 mentions)
Dmem low glucose, by Cytiva (1 mentions)
Cell recovery solution, by BD (1 mentions)
7 protocols using 40 μm nylon mesh
1
Isolating Retinal Microglia from rd10 Mice
2
Single-Cell Omics Profiling Protocols
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The scRNA-seq libraries were prepared using the Chromium Single Cell Gel Bead and 3’ Reagent Kit v2 and v3 (10 × Genomics, Pleasanton, CA). About 15,000–20,000 cells were loaded in the 10 × Genomics Chromium single-cell microfluidics (10 × Genomics, Pleasanton, CA) to generate single-cell gel beads in emulsion (GEMs). After size and quality check with Agilent 2100 Bioanalyzer, the libraries were sequenced on BGISEQ-500 (BGI, China) in PE100 mode.
For scATAC-seq, the fresh protoplasts were lysed with 10 mM Tris–HCL, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 0.1% NP-40, 0.5% Dodecyl Maltoside (DDM), and filtered with a 40-μm nylon mesh (CORNING, USA). The nuclei were then spun down with 500 g for 3 min, resuspended, and purified using SH800S flow cytometer (Sony, Japan). For each sample, about 12,000 nuclei were used for Tn5 tagmentation and amplification following the previous protocol [62 (link)]. The MGISEQ-2000 (BGI, China) with PE50 mode was used for sequencing the libraries.
For scATAC-seq, the fresh protoplasts were lysed with 10 mM Tris–HCL, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 0.1% NP-40, 0.5% Dodecyl Maltoside (DDM), and filtered with a 40-μm nylon mesh (CORNING, USA). The nuclei were then spun down with 500 g for 3 min, resuspended, and purified using SH800S flow cytometer (Sony, Japan). For each sample, about 12,000 nuclei were used for Tn5 tagmentation and amplification following the previous protocol [62 (link)]. The MGISEQ-2000 (BGI, China) with PE50 mode was used for sequencing the libraries.
3
Organoid Culture and Harvest Protocol
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After harvesting from the Matrigel or collagen gel, digestion, and disruption by pipetting 30 times in TrypLE Express solution, organoids were collected by centrifugation at 440×g for 3 min. After washing with basal medium and harvesting by centrifugation, the cells were resuspended with growth medium, filtered through a 40-μm nylon mesh (Corning), and seeded in six-well plates coated with type I collagen or Transwells (all from Corning). The medium was replaced every 2–3 days. Cells were cultured in a 5% CO2 incubator at 37 °C.
4
Isolation and Culture of Rat Vascular Smooth Muscle Cells
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The carotid artery and aorta of adult male Sprague-Dawley rats (9–11 weeks old) were cut longitudinally and the lumen gently rubbed with a cotton swab to remove the endothelium. Vessel segments of 3-5 mm in length were digested in Dulbecco’s modified Eagle’s medium (DMEM; low glucose; HyClone Laboratories, Logan, UT) containing collagenase (type II; 1 mg/ml) for 5 hours at 37°C. Cells were filtered (40 μm nylon mesh; Corning, NY), cultured in DMEM supplemented with 10% FBS (Invitrogen Life Technologies, Grand Island, NY), 2% penicillin-streptomycin, 1% fungizone, epidermal growth factor (25 ng/ml), basic fibroblast growth factor (10 ng/ml) and grown until confluent. Experiments were subsequently performed on 1st/2nd passage VSMCs plated at a density of 125-150 cells/mm2 in DMEM-containing 10% FBS for 24 hours. Thereafter, VSMCs were washed and the media replaced with DMEM supplemented with insulin/transferring/selenium (BD Bioscience, Bedford, MA) for 48 hours. To assess the effect of hyperglycaemia, VSMCs were plated in DMEM containing 5 mM D-glucose for 48 hours and thereafter supplemented with 25 mM D-glucose (Sigma, St-Louis MO), 30 mM L-glucose (Sigma) or 30 mM mannitol (Sigma) for 24 or 48 hours. DNA and protein synthesis was determined by 3H-thymidine and 3H-leucine uptake respectively, as previously described [10 (link)].
5
Isolation and Differentiation of Murine T Cells
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Before in vitro T cell differentiation, splenic single cell suspensions were generated by passing spleens through a 40 μm nylon mesh (Corning), followed by a washing step in R10+ medium (see Table 1 ). Erythrocytes were removed in 5 ml ACK-Lysis-Buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) for 5 min. Thereafter, the cell suspension was washed in R10+ medium and cell number was determined. Isolation of naïve T cells was performed with mouse CD4+ T Cell Isolation and CD25 Isolation Kits (Miltenyi Biotec) according to manufacturer's instructions. For T cell differentiation, 1 × 106 CD4+CD25− T cells were seeded in 48-well plates coated with anti-CD3/anti-CD28 antibodies (BioXcell, 10 μg/ml each). The differentiation of the respective T cell population was induced by stimulating the cells according to Table 1 for 5 days.
6
Isolation of Atrial Cardiomyocytes from Transgenic Mice
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Hearts were removed from heterozygous and homozygous DctCre::Z/EG mice as previously described (Levin et al. 2009 (link); Hwang et al. 2014 ). The atria were then dissected away from the atrioventricular annulus under a dissecting microscope, rinsed free of blood in PBS (phosphate buffered saline) and incubated in 0.25% trypsin at 37°C for 30 min. Single cells were isolated from the atria by repeated, gentle pipetting in PBS containing 10% FBS (fetal bovine serum), followed by filtration through a 40-μm nylon mesh (Corning Life Sciences, Tewksbury, MA). The cells were then washed with PBS × 3 and placed in melanocyte isolation media for 6–8 h. Unattached myocardial cells, including myocytes, were rinsed away and patch clamp recordings were obtained from green fluorescent cells the next day. G418 (Invitrogen) at a concentration of 100 μg/mL was added to the culture medium to suppress fibroblast growth.
7
Organoid Culture and Characterization
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After harvesting from Matrigel, digestion, and disruption by pipetting 30 times in TrypLE Express solution, organoids were collected by centrifugation at 440 × g for 3 min. Alternatively, after treated with cell recovery solution (BD Biosciences) for 30 min on ice, organoids were harvested by centrifugation at 440 × g for 3 min and disrupted in 1 mL of basal medium by passing through a 26-gauge needle 10 times, followed by centrifugation at 440 × g for 3 min. After washing with basal medium and harvest by centrifugation, cells were resuspended with human organoid culture medium, filtered through a 40 μm nylon mesh (Corning), and seeded on type I collagen-coated 6-well plates, 12-well plates, or Transwells. The medium was replaced every 2–3 days. Cell confluence was determined by microscope observation (6- or 12-well plates) or induction of TEER values (Transwell). To determine CYP3A4 induction upon ligand treatment and SGLT1 activity, the medium was replaced with differentiation medium [Advanced DMEM/F-12 with 12.5% RN CM, 10 mM HEPES (pH 7.3), 1% BSA, 2 mM GlutaMAX I, 50 ng/mL mEGF, 10 μM Y-27632, 500 nM A83-01, 100 μg/mL gentamicin, 100 units/mL penicillin, and 100 μg/mL streptomycin] and cultured in a 5% CO2 incubator at 37°C. The cells were used for assays when they reached confluency.
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