TITLE: 3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe
AUTHORS: Helen Graves, Steven Evans, Michael Fauler, Manfred Frick, Sterghios A. Moschos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 preclinical 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</span><span style = "font-style:italic;">. </span><span>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.</span></div></div>

[STEPS]
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Rinse the fresh, perfused mouse lung, with sterile PBS to eliminate any blood.
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Transfer the lung lobe to  in a sterile tissue vial (ensure full tissue submersion) and keep  until all lungs are harvested.
[digestion buffer]
on ice
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Transfer tissue into a gentleMACS C tube using a sterile spatula and add  per tube (see Notes 6 and 7).
[Collagenase Buffer]
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Close the gentleMACS C tube using the provided impeller cap (see Note 8).
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Mix by gentle inversion three times.
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Keep  until all lungs are prepared.
on ice
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Once all the lungs for your experiment are prepared, process them sequentially on the gentleMACS tissue dissociator by placing the tubes, inverted, onto the device, and running program m_lung_01.01 provided by the supplier, to disrupt the tissue.
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Transfer the gentleMACS C tubes onto an oscillating platform incubator set at  and shake at .
37 °C
33
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Return the gentleMACS C tubes onto the gentleMACS tissue dissociator and process the samples using program m_lung_02.01 to achieve monocellular suspensions.
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Pulse-centrifuge the gentleMACS C tubes at  to collect cells at the bottom of the tube.
Centrifuge: 400 34, 10 s
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Assemble a 50 ml centrifugation tube per lung lobe with a 40 μm cell strainer.
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Pass the cells through the 40 μm cell strainer using a 5 ml syringe plunger.
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Rinse the strainer with 3 ×  into the 50 ml centrifugation tube.
[digestion buffer]
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
With the strainer still attached to the centrifuge tubes, centrifuge the cells at , to improve cell recovery rate.
Centrifuge: 400 34, 10 min, 4 10
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Carefully remove and discard the supernatant without disturbing the cell pellet.
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Resuspend the cells in  by gentle mixing using a 25 ml serological pipette, and keep on wet ice or at .
[cell resuspension buffer]
4 °C
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Add ; quickly cap the tube and invert to mix.
[deionized water]
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
After  make the solution isotonic by adding  (see Note 9).
[3.6% w/v sodium chloride]
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Centrifuge the cells at .
Centrifuge: 400 34, 10 min, 4 10
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Carefully remove and discard the supernatant without disturbing the cell pellet.
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Resuspend the cells in .
[cell resuspension buffer]
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Count your cells, for example, by trypan blue exclusion assay on a hemocytometer (mix 25 μl of Trypan Blue with 25 μl of cell suspension and load onto the hemocytometer; count as per hemocytometer manufacturer instructions) or automated cell counting apparatus.
?. [3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe]
Store  or at  until cell sorting is carried out (see Note 10).
on ice
4 °C