Measuring the action of oligonucleotide therapeutics in the lung at the cell type specific level by Tissue Disruption and Cell Sorting (TDCS)

Graves, Helen, Evans, Steve, Fauler, Michael, Frick, Manfred and Moschos, Sterghios (2019) Measuring the action of oligonucleotide therapeutics in the lung at the cell type specific level by Tissue Disruption and Cell Sorting (TDCS). In: Oligonucleotide-based Therapies. Methods in Molecular Biology (2036). Springer, New York, NY, pp. 187-203. ISBN 9781493996698, 9781493996704

[img]
Preview
Text
2019_Protocol_.pdf - Published Version
Available under License Creative Commons Attribution 4.0.

Download (634kB) | Preview
[img] Text
Graves et al - Measuring the action of oligonucleotide therapeutics in the lung AAM.docx - Accepted Version

Download (67kB)
Official URL: https://doi.org/10.1007/978-1-4939-9670-4_11

Abstract

The clinical potential of DNA and RNA-targeting therapeutics for airways disease has been hampered by the poor translation of promising drug candidates from cell culture to in vivo models and the clinic. For example, classical pre-clinical approaches routinely report 20-60% target knockdown effects in the lung, where 1 or 2 log effects are observed in isolated cell cultures in vitro. Preparation of monocellular suspensions of Tissues by mechanoenzymatic Disruption followed by Cell Sorting (TDCS) after in vivo drug dosing, however, can offer pharmacokinetic and pharmacodynamic insights on the effects of drugs to precise cell subpopulations. Moreover, this can be reliably achieved with up to 66% fewer animals than standard in vivo pharmacology approaches due to lower data variance afforded through analytics on defined, viable cell numbers. Here we describe the TDCS methodology for the isolation of total lung epithelia, lung macrophages, and epithelium/macrophage-depleted cell fractions from mouse lungs using a two-stage sorting process of immunomagnetic bead separation followed by flow cytometric sorting using fluorescent antibodies against well-established surface markers such as F4/80, CD11b, and CD326. Validated antibodies for additional cell types and markers are also provided.

Item Type: Book Section
Uncontrolled Keywords: Lung, cell type specific in vivo pharmacology, oligonucleotides, fluorescence activated cell sorting, magnetic cell sorting, RNAi, siRNA, antisense, microRNA
Subjects: B200 Pharmacology, Toxicology and Pharmacy
B800 Medical Technology
B900 Others in Subjects allied to Medicine
C500 Microbiology
Department: Faculties > Health and Life Sciences > Applied Sciences
Depositing User: Paul Burns
Date Deposited: 08 Jan 2019 11:57
Last Modified: 31 Jul 2021 20:15
URI: http://nrl.northumbria.ac.uk/id/eprint/37510

Actions (login required)

View Item View Item

Downloads

Downloads per month over past year

View more statistics