enzymatic dissociation
Recently Published Documents


TOTAL DOCUMENTS

59
(FIVE YEARS 13)

H-INDEX

17
(FIVE YEARS 1)

2022 ◽  
Vol 15 ◽  
Author(s):  
Luoziyi Wang ◽  
Yiwen Qian ◽  
Xin Che ◽  
Jing Jiang ◽  
Jinshan Suo ◽  
...  

Microglia, the primary resident immunocytes in the retina, continuously function as immune system supervisors in sustaining intraocular homeostasis. Microglia relate to many diseases, such as diabetic retinopathy, glaucoma, and optic nerve injury. To further investigate their morphology and functions in vitro, a reliable culture procedure of primary human retinal microglia is necessary. However, the culture condition of microglia from the adult retina is unclear. Researchers created several protocols, but most of them were carried out on rodents and newborns. This study describes a protocol to isolate and characterize human primary retinal microglia from human post-mortem eyes. The whole procedure started with removing the retinal vessels, mechanical separation and enzymatic dissociation, filtration, and centrifugation. Then, we cultured the cell suspensions on a T-75 flask for 18 days and then shook retinal microglia from other retinal cells. We found numerous retinal microglia grow and attach to Müller cells 10 days after seeding and increase rapidly on days 14–18. Iba1 and P2RY12 were used to qualify microglia through immunofluorescence. Moreover, CD11b and P2RY12 were positive in flow cytometry, which helps to discriminate microglia from other cells and macrophages. We also observed a robust response of retinal microglia in lipopolysaccharide (LPS) treatment with proinflammatory cytokines. In conclusion, this study provides an effective way to isolate and culture retinal microglia from adult human eyes, which may be critical for future functional investigations.


Author(s):  
Madison Trujillo ◽  
Taylor McElroy ◽  
Taurean Brown ◽  
Pilar Simmons ◽  
Fabio Ntagwabira ◽  
...  

2021 ◽  
Author(s):  
Jongeun Park ◽  
Veronika Kedlian ◽  
Chenqu Suo ◽  
Liam Bolt ◽  
Alexander Steemers ◽  
...  

This Protocol is intended for human Thymus single cell dissociation. It includes tissue preservation and handling, Enzymatic dissociation, FACS an MACS enrichments. Developed In the Teichmann lab at the Sanger institute, Wellcome Gemone Campus, UK VK, CS and JP contributed equally


Membranes ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 687
Author(s):  
Nathalia Barth de Oliveira ◽  
Ana Carolina Irioda ◽  
Priscila Elias Ferreira Stricker ◽  
Bassam Felipe Mogharbel ◽  
Nádia Nascimento da Rosa ◽  
...  

Adipose tissue-derived mesenchymal stem cells (ADMSCs) are promising candidates for regenerative medicine, as they have good cell yield and can differentiate into several cell lines. When induced to the neuronal differentiation, they form neurospheres composed of neural precursors (NPs) that can be an alternative in treating neurodegenerative diseases. This study aimed to characterize NPs from neurospheres obtained after seeding ADMSCs on a natural polyisoprene-based membrane. The ADMSCs were isolated from adipose tissue by enzymatic dissociation, were subjected to trilineage differentiation, and were characterized by flow cytometry for specific ADMSC surface markers. For neuronal differentiation, the cells were seeded on polystyrene flasks coated with the membrane and were characterized by immunocytochemistry and RT-PCR. The results demonstrated that the isolated cells showed characteristics of ADMSCs. At 15 to 25 days, ADMSCs seeded on the natural membrane developed neurospheres. Then, after dissociation, the cells demonstrated characteristic neuronal markers expressed on NPs: nestin, ß-III tubulin, GFAP, NeuN, and the YAP1/AMOT in the cytoplasm. In conclusion, it was demonstrated that this membrane differentiates the ADMSCs to NPs without any induction factors, and suggests that their differentiation mechanisms are related to mechanotransduction regulated by the YAP and AMOT proteins.


2021 ◽  
Author(s):  
Atsuko Miyawaki‐Kuwakado ◽  
Qianmei Wu ◽  
Akihito Harada ◽  
Kosuke Tomimatsu ◽  
Takeru Fujii ◽  
...  

2021 ◽  
Vol 11 (1) ◽  
pp. 92-99
Author(s):  
V. N. Pavlov ◽  
A. A. Kazikhinurov ◽  
R. A. Kazikhinurov ◽  
M. A. Agaverdiev ◽  
I. F. Gareev ◽  
...  

Stromal vascular fraction (SVF) is a heterogeneous cell extract obtained with enzymatic dissociation of adipose tissue followed by centrifugation. This population includes many different cell types, i.a. adipose tissue stem cells (ATSCs), vascular endothelial and smooth muscle cells and their precursors, pericytes, fibroblasts, macrophages, T-lymphocytes, etc., excluding mature adipocytes. The main SVF component is ATSCs capable of self-renewal and multipotent differentiation. Since early research on SVF, an extensive effort has been aimed at understanding its clinical applications promoting a significant progress in the SVF use for treatment of various diseases and injuries. The past decade has witnessed an upward publication trend in basic and clinical research into the SVF therapeutic value. Manifold methods and devices for the SVF isolation from human liposuction lipoaspirate have been developed, continuously contributing to preclinical and clinical trials of its safety and efficacy. This review discusses the main properties and functions of the SVF cell population, its efficacy and safety for human therapy.


2020 ◽  
Author(s):  
Samuel E. Marsh ◽  
Tushar Kamath ◽  
Alec J. Walker ◽  
Lasse Dissing-Olesen ◽  
Timothy R. Hammond ◽  
...  

AbstractA key aspect of nearly all single cell experiments is the necessity to dissociate intact tissues into single cell suspensions for processing. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression changes during this process. Microglia, the brain’s resident macrophages, are a highly dynamic population that are extremely sensitive to their microenvironment and have been shown to dramatically alter their transcriptome upon stimulation. We demonstrate that use of enzymatic dissociation methods on mouse central nervous system (CNS) tissue induces an aberrant gene expression signature in microglia that can significantly confound downstream analysis. To minimize this issue, we developed a flexible protocol, that can be used with existing enzymatic protocols for fresh tissue, to eliminate artifactual gene expression while allowing for increased cell type diversity and yield. We demonstrate efficacy of this protocol in analysis of diverse CNS cell types and sorted myeloid populations while using enzymatic dissociation. Generation of new and reanalysis of previously published human brain single nucleus RNAseq (snRNA-seq) datasets reveal that a similar signature is also present in post-mortem tissue. Through novel snRNA-seq analysis of acutely-resected neurosurgical tissue we demonstrate that this signature can be induced in human tissue due to technical differences in sample processing. These results provide key insight into the potential confounds of enzymatic digestion and provide a solution to allow for enzymatic digestion for scRNA-seq while avoiding ex vivo transcriptional artifacts. Analysis of human tissue reveals potential for artifacts in current and future snRNA-seq datasets that will require deeper analysis and careful consideration to separate true biology from artifacts related to post-mortem processes.


Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Michael Klein ◽  
Robert Geiger ◽  
Mori J Krantz ◽  
Robert Goldstein ◽  
Thomas P Flagg ◽  
...  

Introduction: Methadone is the second most frequently reported cause of drug-induced cardiac arrest in pharmacovigilance databases, yet the mechanism of its pro-arrhythmia is unclear. Methadone-induced QTU wave prolongation has been repeatedly observed and attributed to inhibition of the delayed rectifier hERG current, but QTU fusion suggests the inwardly rectifying K + current (IK1) might also be affected by methadone. Hypothesis: Methadone pro-arrhythmia is associated with potent block of the IK1 current responsible for rapid terminal repolarization of the cardiac action potential (AP). Methods: Human Kir2.2, encoding the IK1 current, was transiently expressed in COS cells. hERG1a was stably expressed in CHO cells. Cardiac myocytes from swine were obtained by ventricular enzymatic dissociation. Ionic current and APs were measured using patch-clamp methods. Methadone HCl (R+S racemates) was dissolved in Tyrode solution. Results: Methadone suppressed IK1 current with an IC 50 of 1.47 uM (Fig 1A). Methadone also suppressed outward IK1 (-60 mV) measured in swine myocytes (Ba 2+ -sensitive current) with an IC 50 of 1.52 μM. Methadone suppressed hERG currents with an IC 50 of 2.1 μM. APs measured in swine myocytes exhibited significant prolongation (13 ± 4 % increase of APD 90 , p<0.029, n=7) as well as slowing of the rate of terminal repolarization (a specific marker of IK1 blockade) in the presence of 1 μM methadone (Fig. 1B). Fluctuations of diastolic voltage increased by 30 ± 12 and 151 ± 27 % (n=3; p<0.04) in 0.1 and 1 μM methadone, respectively, consistent with a reduction in membrane stability. Conclusions: Methadone is an equipotent blocker of IK1 and hERG. The effect of IK1 block coupled with modest hERG block has a synergistic effect on terminal repolarization that may partially explain the pro-arrhythmic impact of methadone. Moreover, this observation may be generalized to other drugs where unsuspected IK1 blockade may contribute to pro-arrhythmia and torsade de pointes.


BioTechniques ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 388-391
Author(s):  
Karla P Garcia-Pelagio ◽  
Stephen JP Pratt ◽  
Richard M Lovering

Isolated myofibers are commonly used to understand the function of skeletal muscle in vivo. This can involve single isolated myofibers obtained from dissection or from enzymatic dissociation. Isolation via dissection allows control of sarcomere length and preserves tendon attachment but is labor-intensive, time-consuming and yields few viable myofibers. In contrast, enzymatic dissociation is fast and facile, produces hundreds of myofibers, and more importantly reduces the number of muscles/animals needed for studies. Biomechanical properties of the sarcolemma have been studied using myofibers from the extensor digitorum longus, but this has been limited to dissected myofibers, making data collection slow and difficult. We have modified this tool to perform biomechanical measurements of the sarcolemma in dissociated myofibers from the flexor digitorum brevis.


Sign in / Sign up

Export Citation Format

Share Document