Protocols for endothelial cell isolation from mouse tissues: kidney, spleen, and testis

Dumas, Sébastien J.; Meta, Elda; Conchinha, Nadine V.; Sokol, Liliana; Chen, Rongyuan; Borri, Mila; Teuwen, Laure-Anne; Veys, Koen; García-Caballero, Melissa; Geldhof, Vincent; Treps, Lucas; Zeeuw, Pauline de; Falkenberg, Kim D.; Dubois, Charlotte; Parys, Maciej; Rooij, Laura P.M.H. de; Rohlenová, Kateřina; Goveia, Jermaine; Schoonjans, Luc; Dewerchin, Mieke; Eelen, Guy; Li, Xuri; Kalucka, Joanna; Carmeliet, Peter

doi:10.1016/j.xpro.2021.100523

Cited by 9 publications

(8 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CD31 + cells retained in the LS column were eluted with PEB buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) and centrifuged at 300 g for 5 minutes. CD45 – CD31 + cells corresponding to endothelial cells were directly pelleted for RNA or protein isolation ( 71 – 73 ).…”

Section: Methodsmentioning

confidence: 99%

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity

Bartoli¹,

Debant²,

Chuntharpursat‐Bon³

et al. 2022

Journal of Clinical Investigation

View full text Add to dashboard Cite

Piezo1 forms mechanically-activated non-selective cation channels that contribute to endothelial response to fluid flow. Here we reveal an important role in the control of capillary density. Conditional endothelial-specific deletion of Piezo1 in adult mice depressed physical performance. Muscle microvascular endothelial cell apoptosis and capillary rarefaction were evident and sufficient to account for the effect on performance. There was selective upregulation of thrombospondin-2 (TSP2), an inducer of endothelial apoptosis, with no effect on thrombospondin-1 (TSP1), a related important player in muscle physiology. TSP2 was poorly expressed in muscle endothelial cells but robustly expressed in muscle pericytes, in which nitric oxide (NO) repressed the Tsp2 gene without effect on Tsp1. In the endothelial cells, Piezo1 was required for normal expression of endothelial nitric oxide synthase (eNOS). The data suggest an endothelial-pericyte partnership of muscle in which endothelial Piezo1 senses blood flow to sustain capillary density and thereby maintain physical capability.

show abstract

Section: Methodsmentioning

confidence: 99%

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity

Bartoli¹,

Debant²,

Chuntharpursat‐Bon³

et al. 2022

Journal of Clinical Investigation

View full text Add to dashboard Cite

show abstract

“…In addition, in this method we used a new combination of digestive enzymes including collagenase I, collagenase II, collagenase IV, DNase I, and hyaluronidase in order to maximize the resulting amount of single cells from the biopsy sample. Other published studies, in human or mice, used either collagenase I, collagenase II, or collagenase I combined with DNase I ( Dumas et al, 2021 ; Zhang et al, 2021 ; Zimmerman et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

Isolation of Viable Single Cells With High Yield and Purity Using a Small Amount of Human Kidney Tissue Biopsy

Yaigoub

Tirichen

Xin

et al. 2022

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

Objective: Establishment of an efficient method of preparing human kidney single cell suspension, using a very small amount of tissue puncture.Methods: Samples of human kidney tissue puncture were cut into pieces, and then 80 μL of the digestive enzyme were added to each punctured tissue to induce enzymatic digestion. The enzyme combination is composed of collagenases, DNase and hyaluronidase and the sample was incubated 20 min at 37°C. The obtained cell suspension was filtered through a 70 μm cell strainer, centrifuged at 300 g for 5 min and the supernatant was removed, then the pellet was resuspended in 3 ml of DMEM (Dulbecco’s Modified Eagle’s Medium). Cell suspension was sorted and purified by flow sorting to remove dead cells and obtain a cell suspension with higher viability rate.Results: We found that 1) diverse single cells of human kidney can be obtained by the digestive enzyme, as observed under the light microscope, with different sizes, normal cell morphology and good dispersion. 2) (2-3) × 106 single cells can be extracted from one fresh punctured kidney tissue of about 10 mg, with a cell viability rate of more than 80%.Conclusion: In this work we generated a comprehensive and high-resolution single-cell method, which is simple and efficient for preparing single cell suspension from a minimal amount of human kidney tissue. This method can facilitate the study of renal cell biology and the pathogenesis of kidney diseases.

show abstract

“…We followed a preexisting protocol 26 , 27 with some modifications. In brief, 4 h after the thioglycollate challenge, mice were perfused with ice-cold PBS transcardially until the colorless fluid was observed coming from the right atrium.…”

Section: Methodsmentioning

confidence: 99%

NLRP3 inflammasome activation in neutrophils directs early inflammatory response in murine peritonitis

Fukui

Bruggen

et al. 2022

Sci Rep

View full text Add to dashboard Cite

NLR family pyrin domain containing 3 (NLRP3) inflammasome mediates caspase-1-dependent processing of inflammatory cytokines such as IL-1β, an essential endothelial activator, and contributes to the pathology of inflammatory diseases. To evaluate the role of NLRP3 in neutrophils in endothelial activation, which is still elusive, we used the thioglycollate-induced peritonitis model characterized by an early neutrophil influx, on Nlrp3−/− and Nlrp3+/+ mice. Nlrp3−/− mice recruited fewer neutrophils than Nlrp3+/+ into the peritoneum and showed lower IL-1β in peritoneal lavage fluid. The higher production of IL-1β in Nlrp3+/+ was neutrophil-dependent as neutrophil depletion prevented the IL-1β production. The Nlrp3+/+ neutrophils collected from the peritoneal fluid formed significantly more filaments (specks) than Nlrp3−/− neutrophils of ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain), a readout for inflammasome activation. Intravital microscopy revealed that leukocytes rolled significantly slower in Nlrp3+/+ venules than in Nlrp3−/−. Nlrp3−/− endothelial cells isolated from mesenteric vessels demonstrated a lower percentage of P-selectin-positive cells with lower intensity of surface P-selectin expression than the Nlrp3+/+ endothelial cells evaluated by flow cytometry. We conclude that neutrophils orchestrate acute thioglycollate-induced peritonitis by producing IL-1β in an NLRP3-dependent manner. This increases endothelial P-selectin expression and leukocyte transmigration.

show abstract

Protocols for endothelial cell isolation from mouse tissues: kidney, spleen, and testis

Cited by 9 publications

References 8 publications

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity

Isolation of Viable Single Cells With High Yield and Purity Using a Small Amount of Human Kidney Tissue Biopsy

NLRP3 inflammasome activation in neutrophils directs early inflammatory response in murine peritonitis

Contact Info

Product

Resources

About