Isolation of Live Leukocytes from Human Inflammatory Muscles

In inflammatory myopathies, the self-reactive immune cells involved in muscle aggression have been studied mostly using histological assessment of muscle biopsy sections; this methodology provides the advantage of visualizing and identifying cells within the tissue, but it does not allow further inv...

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Bibliographic Details
Published in:Methods and protocols 2021-10, Vol.4 (4), p.75
Main Authors: Coudert, Jerome D., McLeish, Emily, Sooda, Anuradha, Slater, Nataliya, Beer, Kelly, Paramalingam, Shereen, Lamont, Phillipa J., Needham, Merrilee
Format: Article
Language:English
Subjects:
Aggression
autoimmunity
Biopsy
Body fat
Cell culture
Centrifugation
Connective tissue
enzymatic digestion
Flow cytometry
immune cells
Inflammation
isolation
Leukocytes
Lymphocytes
Molecular modelling
Myocytes
myopathy
myositis
Protocol
Skeletal muscle
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Summary:In inflammatory myopathies, the self-reactive immune cells involved in muscle aggression have been studied mostly using histological assessment of muscle biopsy sections; this methodology provides the advantage of visualizing and identifying cells within the tissue, but it does not allow further investigation. To gain access to live and isolated cells, many studies utilized blood samples; however, in the absence of biological tools to discriminate the leukocytes associated with the autoimmune process from those that emerged from responses against pathogens, the information observed on circulating immune cells often lacks in specificity, and thus result interpretation may prove difficult. In order to selectively retrieve self-reactive immune cells, we developed a protocol to isolate live leukocytes from human muscle biopsies, which allows for further analysis using a large range of methodologies. The protocol uses enzymatic digestion to release live leukocytes from freshly collected skeletal muscle samples, followed by filtration and separation of the leukocytes from the myocytes by density gradient centrifugation. The isolated cells can be submitted immediately to various analysis strategies to characterize ex vivo the specific cellular and molecular mechanisms responsible for self-directed immune muscle aggression or may be placed in culture for expansion.
ISSN:2409-9279
2409-9279
DOI:10.3390/mps4040075