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.
|Title of host publication||Oligonucleotide-based Therapies|
|Editors||Olof Gissberg, Rula Zain, Karin Lundin|
|Place of Publication||New York, NY|
|Publication status||Published - 2019|
|Name||Methods in Molecular Biology|