3.1 Preparation of Monocellular Lung Suspension from a Mouse Lung Lobe
Helen Graves, Steven Evans, Michael Fauler, Manfred Frick, Sterghios A. Moschos
Lung
Cell type-specific in vivo pharmacology
Oligonucleotides
Fluorescence activated cell sorting
Magnetic cell sorting
RNAi
siRNA
Antisense
MicroRNA
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 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 . 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.
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.
Transfer the lung lobe to 2mL in a sterile tissue vial (ensure full tissue submersion) and keep On ice until all lungs are harvested.
Transfer tissue into a gentleMACS C tube using a sterile spatula and add 5mL per tube ( see Notes 6 and 7 ).
Close the gentleMACS C tube using the provided impeller cap ( see Note 8 ).
Mix by gentle inversion three times.
Keep On ice until all lungs are prepared.
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.
Transfer the gentleMACS C tubes onto an oscillating platform incubator set at 37°C and shake at 250rpm.
Return the gentleMACS C tubes onto the gentleMACS tissue dissociator and process the samples using program m_lung_02.01 to achieve monocellular suspensions.
Pulse-centrifuge the gentleMACS C tubes at 400x g to collect cells at the bottom of the tube.
Assemble a 50 ml centrifugation tube per lung lobe with a 40 μm cell strainer.
Pass the cells through the 40 μm cell strainer using a 5 ml syringe plunger.
Rinse the strainer with 3 × 1mL into the 50 ml centrifugation tube.
With the strainer still attached to the centrifuge tubes, centrifuge the cells at 400x g,4°C, to improve cell recovery rate.
Carefully remove and discard the supernatant without disturbing the cell pellet.
Resuspend the cells in 4mL by gentle mixing using a 25 ml serological pipette, and keep on wet ice or at 4°C.
Add 20mL; quickly cap the tube and invert to mix.
After 0h 0m 20s make the solution isotonic by adding 8mL ( see Note 9 ).
Centrifuge the cells at 400x g,4°C.
Carefully remove and discard the supernatant without disturbing the cell pellet.
Resuspend the cells in 5mL.
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.
Store On ice or at 4°C until cell sorting is carried out ( see Note 10 ).
