2025-12-31
ENA first public
ERC000011
ENA-CHECKLIST
Department of Internal Medicine (Nephrology) & Einthoven Laboratory of Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands
INSDC center name
public
INSDC status
ERS22615981
SRA accession
E-MTAB-14703:SI-NA-C1
Submitter Id
28mo
day
age
ArrayExpress
broker name
endothelial cell
cell_type
not collected
collection date
house mouse
common name
Protocols: TISSUE COLLECTION C57Bl/6j mice were used for endothelial cell (EC) isolation across various tissues and life stages. The embryonic days were counted starting from the day of detection of vaginal plug on the pregnant females, considered as E0.5. For brain ECs, 62 embryos (from 8 females) were used at E13.5, 41 embryos (from 5 females) at E15.5, and 38 embryos (from 5 females) at E18.5. For heart ECs, 38 embryos (from 5 females) were used at E13.5, 41 embryos (from 5 females) at E15.5, and 38 embryos (from 5 females) at E18.5. Liver ECs were isolated from 38 embryos (from 5 females) at E13.5, 41 embryos (from 5 females) at E15.5, and 19 embryos (from 5 females) at E18.5. Kidney and lung ECs were obtained from 90 embryos (from 12 females) at E13.5, 59 embryos (from 7 females) at E15.5, and 38 embryos (from 5 females) at E18.5. Embryos were collected upon cervical dislocation of the pregnant females at timepoint E13.5, E15.5, E18.5, and brain, heart, kidneys, liver, and lungs were obtained by dissection and kept on ice-cold HBSS. At P1, 21 pups (from 7 females) were used for all organ ECs. From P14 onward, EC isolations were performed from 8 male mice. Organs were dissected from pups upon decapitation and kept on ice-cold HBSS. Adult mice were anesthetized with an intraperitoneal injection of ketamine/xylazine (Dechra, Innovet) and blood was collected from the right ventricle. Mice were then submitted to left heart perfusion with 3ml PBS (1ml/min), and brain, heart, kidneys, liver, lungs, and skeletal muscles (soleus, tibialis anterior, extensor digitorum longus) were collected on ice-cold HBSS.  TISSUE DISSOCIATION FOR EC ISOLATION ECs were isolated using tissue- and stage-specific protocols, optimized from previously published protocols (Dumas et al. Star Protocols 2021, Conchinha et al. Star Protocols 2021, Sokol et al. Star Protocols 2021).  For brain ECs, the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, 130-092-628) was used. At all timepoints, starting from mechanically minced brain tissue, digestion was performed sequentially at 37°C with enzyme mix 1 (Buffer X and Enzyme P) for 17 minutes with slow rotation, and followed by the addition of enzyme mix 2 (Buffer Y and Enzyme A) with mechanical dissociation by pipetting with a Pasteur pipette. Samples are further incubated for 12 minutes at 37°C with slow rotation, and then sheared through a 20G needle 10 times. After an extra incubation of 10 minute at 37°C, digestion was stopped with ice-cold HBSS, and the samples were filtered through a 70 μm cell strainer and centrifuged at 300g for 5 min (repeated twice for the supernatants). Myelin removal was performed using Myelin Removal Magnetic Microbeads (Miltenyi Biotec, 130-096-733) and QuadroMACS separators with LS columns (Miltenyi Biotec). CD31+ cells were enriched with CD31 Microbeads (Miltenyi Biotec, 130-097-418) before flow cytometry analysis and sorting. For heart ECs, mechanically minced tissues were digested in a solution containing 1 mg/ml collagenase II (Gibco, 17101-015), 2.5 mg/ml collagenase IV (Worthington, LS004188), and 27 IU/ml DNase I (Sigma-Aldrich, D4527) in KnockOut™ DMEM (Gibco, 10829018), supplemented with penicillin/streptomycin (Thermo Fisher Scientific, 15140122), 2× Antibiotic-Antimycotic (Thermo Fisher Scientific, 15240062), 1 mM sodium pyruvate (Thermo Fisher Scientific, 1360070), and 1× MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific, 11140035). Incubation times were adjusted by developmental stage: 35 minutes at E13.5 and E15.5, 40 minutes at E18.5, and 45 minutes at P1. Digestion was stopped by adding ice-cold buffer (0.1%BSA, 2mm EDTA in PBS). Samples were filtered through a 40 μm strainer, centrifuged, and enriched for CD31+ cells using magnetic beads (except for E13.5 timepoint), before sorting. For later timepoints, tissues were incubated 25 minutes at 37°C in the same digestion buffer, while shaking every 5 minutes, further homogenized in gentleMACS C tubes (program C; Miltenyi Biotec) and pipetted with 25ml, 10ml and 5ml pipets until well homogenized. Digestion was stopped by adding ice-cold buffer (0.1%BSA, 2mm EDTA in PBS), and sample were filtered on a 100 μm cell strainer and centrifuged twice for the supernatant. After a third centrifugation, CD31+ cells were enriched with CD31 Microbeads before flow cytometry analysis and sorting. For kidney ECs, digestion was performed with 2 mg/ml collagenase I (Sigma-Aldrich, C9891) and 27 IU/ml DNase I (Sigma-Aldrich, D4527) starting from mechanically minced tissue. Incubation times were 20 minutes at E13.5, 40 minutes at E15.5 and later timepoints, shaking every 10 minute. Digestion was stopped with ice-cold buffer (0.1%BSA, 2mm EDTA in PBS) and samples were filtered through a 70 μm strainer (for pup and adult timepoints), then a 40 μm strainer and centrifuged; with CD31+ enrichment carried out before sorting for samples from E18.5 timepoint onwards, before flow cytometry analysis and sorting.   Liver ECs were isolated using a buffer containing 1.25 mg/ml collagenase I (Thermo Fisher Scientific, 17018029), 1.25 mg/ml collagenase II (Thermo Fisher Scientific, 17101015), 0.5 IU/ml dispase (Thermo Fisher Scientific, 171055041), and 67.5 IU/ml DNase I (Sigma-Aldrich, D4527) in KnockOut™ DMEM, supplemented as described for heart tissue. Incubation was performed for 12 minutes at E13.5, 20 minutes at E15.5 and E18.5, shaking every 10 minutes. For pup and adult timepoints, 1ml of digestion buffer was injected in different regions of the livers followed by a 30-minute incubation at 37°C, shaking every 10 minute. Digestion was stopped with ice-cold buffer (0.1% BSA, 2 mM EDTA in PBS), followed by filtration through a 100 μm strainer for pup and adult timepoints, then through a 40 μm strainer, centrifugation, and enrichment for CD31+ cells with magnetic beads before flow cytometry analysis and sorting.  For lung ECs, starting from separated lung lobes, digestion employed 1 mg/ml collagenase II, 2.5 mg/ml collagenase IV, and 20.25 IU/ml DNase I in KnockOut™ DMEM, supplemented as described for heart tissue. Incubation was set to 30 minutes at E13.5, pipetting every 10 minute, followed by mechanical homogenization using gentleMACS Dissociator System (Miltenyi Biotec) with m_lung_02 program. E15.5 samples were incubated for 15 minutes at 37°C, pipetting every 10 minute, then submitted to m_lung_01 program, incubated again with similar pipetting for 15 minutes, and further homogenized with m_lung_02 program. From E18.5 onwards, samples were directly submitted to m_lung_01 program in the digestion buffer, incubated for 30 minutes at 37°C with pipetting every 10 minutes, and further homogenized with m_lung_02 program. Digestion was stopped with ice-cold buffer (0.1% BSA, 2 mM EDTA in PBS), samples were filtered through a 40 μm strainer, and centrifuged. CD31+ cells were enriched with CD31 Microbeads before flow cytometry analysis and sorting for all timepoints except for E13.5 before flow cytometry analysis and sorting. Finely minced skeletal muscles were incubated in digestion buffer made of 1.2 IU/mL dispase (Thermo Fisher Scientific, 171055-041), 2 mg/mL collagenase IV (Worthington, LS004188), 2 mM CaCl₂, and PBS, at 37°C for 45 minutes with pipetting every 15 minutes using a Pasteur pipette. Digestion was stopped by adding ice-cold FACS buffer (0.1% BSA, 2 mM EDTA in PBS). The samples were filtered sequentially through a 70 μm cell strainer and a 40 μm cell strainer. Samples were centrifuged at 300g for 20 minutes, and the resulting pellets were resuspended for CD31+ enrichment. CD31+ cells were isolated using CD31 Microbeads (Miltenyi Biotec, 130-097-418) and QuadroMACS separators with MS columns (Miltenyi Biotec), followed by centrifugation at 300g for 5 minutes. Finally, the enriched CD31+ cell population was stained and prepared for flow cytometry analysis and sorting.  FACS SORTING OF ENDOTHELIAL CELLS For ECs from brain, heart, kidney, lung and muscle, the cell suspensions obtained after organ digestion were incubated for 30min at 4°C with: CD45 (1/100, 561047, BD Biosciences), CD102 (1/200, 105609, BioLegend), CD31 (1/50, 130-111-355, Miltenyi Biotec) and viability dye (1/1000, 65-0865-14, Thermo Fisher Scientific). OneComp ebeads (01–1111–42; Thermo Fisher Scientific) and a 50:50 mix of live and cooked cells (20 minutes at 60°C) were used for compensation. Viable CD45-CD31+ ECs were FACS-sorted using a FACS Aria III (BD Biosciences) and collected in wash buffer (0.1% BSA in PBS). Cells were centrifuged for 6min at 400xg and processed for nuclei isolation. For ECs from liver, the cell suspensions obtained after organ digestion were incubated for 30min at 4°C with: CD45 (1/100, 561047, BD Biosciences), CD102 (1/200, 105609, BioLegend), CD11b (1/500, 101207, BioLegend), CD31 (1/50, 130-111-355, Miltenyi Biotec) and viability dye (1/1000, 65-0865-14, Thermo Fisher Scientific). OneComp ebeads (01–1111–42; Thermo Fisher Scientific) and a 50:50 mix of live and cooked cells (20 minutes at 60°C) were used for compensation. Viable CD11b-CD31+ were FACS-sorted using a FACS Aria III (BD Biosciences) and collected in wash buffer (0.1% BSA in PBS). Cells were centrifuged for 6min at 400xg and processed for nuclei isolation.  NUCLEI ISOLATION FACS-sorted ECs were subjected to nuclei isolation using the low-input protocol from 10x Genomics (CG000169, Rev D). Briefly, up to 100,000 FACS-sorted ECs were centrifuged at 300xg for 5 min at 4°C in 50 µL of PBS containing 0.04% BSA. After centrifugation, 45 µL of supernatant was replaced with chilled lysis buffer (10 mM Tris-HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl₂; 0.1% Tween-20; 0.1% Nonidet P40 Substitute; 0.01% Digitonin; 1% BSA; nuclease-free water). The sample was mixed gently by pipetting three times and incubated on ice for 5 min. Next, 50 µL of dilution buffer (composition identical to the lysis buffer but without Nonidet P40 Substitute and Digitonin) was added, and the sample was centrifuged at 500xg for 5 min at 4°C. The nuclei pellet was resuspended in 45 µL of 1X Nuclei Buffer (PN-2000153, 10x Genomics) and centrifuged again at 500xg for 5 min at 4°C. Finally, the nuclei were resuspended in 7 µL of 1X Nuclei Buffer and processed for nuclei counting and downstream scATAC-seq library preparation. Nucleic acid extraction was performed in single nuclei using the Chromium Single Cell ATAC Reagent Kit (v1) and Chromium Single Cell E chip Kit after a 1h transposition step, according to manufacturer’s instructions (10X Genomics).  The endothelial nuclei were converted to barcoded scATAC-seq libraries using the Chromium Single Cell ATAC Reagent Kit (v1) and Chromium Single Cell E chip Kit (10X Genomics). Samples were processed with Chromium i7 sample index Plate N, Set A. Nuclei from each sample were treated with the same master mix and in the same reaction vessel, by processing every single sample in a single well of a PCR plate.
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
heart
organism part
Mus musculus
scientific_name
male
sex
C57Bl/6j
strain
SI-NA-C1
title