TITLE: 3.2 Isolation of Specific Cell Types from Digested Lung Tissue
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]
?. [3.2 Isolation of Specific Cell Types from Digested Lung Tissue]
Due to the high intrinsic autofluorescence of tissue resident macrophages which can make flow cytometric analysis more challenging and limit the choice of fluorochromes, a two-stage separation protocol is advised whereby macrophages and other myeloid cells are first removed in bulk using CD11b-specific paramagnetic beads, followed by flow cytometric staining and cell sorting (see Note 11; Fig.1).There are a large range of kits and reagents commercially available for the immune-magnetic isolation or depletion of particular cell types in a blood or tissue sample. Magnetic microbeads are coupled to antibodies specific for cell surface epitopes and when combined with a magnetic field, the desired cell type can be isolated or undesired cell types removed. This section describes the use of Miltenyi MACS reagents and equipment but the methods described could be carried out with equivalent reagents from other sources. Similarly, the fluorochromes used can be adapted to the capability of the cell sorter and fluorescent antibodies are available from a wide range of commercial suppliers. Table 1 summarizes antibody suppliers which have proven reliable in our hands when applying TDCS in pharmacological studies (all reagents were used at the manufacturer’s recommended concentration).SpeciesTarget and cloneSupplier and cat. no.ApplicationMouseCD105; 209701BD Biosciences, R and D Systems FAB1320AEndothelial cells or activated macrophages, positive selection; can discriminate the two populations by granularityMouseCD45; 30-F11BD Biosciences 557659, 557235Type I epithelial cell negative selection; Leukocyte positive selection; Macrophage cell positive selection (±)MouseF4/80; BM8Invitrogen MF48021Positive selection of macrophages in a CD11b− population; F4/80 negative selects for neutrophilsMouseCD144; 55-7H1BD Biosciences 560411Endothelial cell positive selectionMouseCD326; G8.8Cambridge Biosciences 118205, Biolegend 118205Epithelial cell positive selection in a CD45−, CD11b− populationMouseCD11b; M1/70Miltenyi Biotech 130-049-601Paramagnetic bead positive selection of macrophages, neutrophils, NK cells, granulocytes and dendritic cells (requires F4/80+ downstream for macrophage selection) and negative selection for epithelial cells (requires CD45−, CD326+ downstream)RatATP-binding cassette subfamily A member 3; 3C9Abcam ab24751Alveolar type II epithelial cells, positive selection (a)RatEpCAM; polyclonalAbcam ab71916Epithelial cells, positive selection (a)RatCD45; OX1eBioscience 12-0461Negative selection for epithelial enrichment (b)RatCD68; ED1Abcam ab31630Macrophages, positive selection (b)RatCD45; REA450Miltenyi Biotec 130-109-682Paramagnetic bead negative selection for epithelial enrichment against leukocytes and macrophages (b)Mouse, ratCD90.1; His51Miltenyi Biotec 130-094-523Paramagnetic bead positive selection for T cells (b)HumanTLR2; TL2.1Invitrogen 16203Positive selection for epithelial cells (b)
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
Retain at least  and store at . Run the remaining sample through the autoMACS Pro as described below.
[each digested lung sample]
4 °C
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
Spin digested lung sample(s) at  to pellet the cells and resuspend them in autoMACS running buffer at 10E7 cells per 80 μl.
Centrifuge: 400 34, 5 min
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
Pass the sample through a filter to ensure a single cell suspension.
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
Add  per 10E7 cells and incubate at  for  (see Note 12).
[CD11b-specific microbeads]
4 °C
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
If not done previously during the incubation, turn on and prime the autoMACS machine and ensure prechilled collection blocks are removed from the fridge.
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
Wash the samples with  and spin at .
[ice-cold autoMACS running buffer]
Centrifuge: 400 34
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
Discard the supernatant and resuspend in .
[autoMACS running buffer]
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
Run samples on the autoMACS, using the “deplete_s” program followed by a quick wash between each sample.
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
Both the CD11b+ and CD11b− fraction are collected into the prechilled collection block.
?. [3.2.1 Separation of CD11b+ Cells Using autoMACS Pro]
Spin samples down at  and resuspend in  (see Note 13; see Fig. 2 for a typical forward scatter/side scatter (FSC/SSC) plot of the CD11b+ and CD11b− fractions).
Centrifuge: 400 34
[stain buffer]
?. [3.2.2 Fluorescent Staining for Flow Cytometric Analysis and Sorting]
Keep aside sufficient cells to set up appropriate controls as exemplified in Fig. 3 (see Notes 14–16).
?. [3.2.2 Fluorescent Staining for Flow Cytometric Analysis and Sorting]
Stain the fractions of CD11b− cells with anti-CD45 and anti-CD326 to identify leukocytes and epithelial cells (see Fig. 1).
?. [3.2.2 Fluorescent Staining for Flow Cytometric Analysis and Sorting]
Stain the fractions of CD11b+ cells with F4/80 to discriminate macrophages.
?. [3.2.2 Fluorescent Staining for Flow Cytometric Analysis and Sorting]
Use  in a total volume of 100 μl cell resuspension buffer.
[each antibody]
?. [3.2.2 Fluorescent Staining for Flow Cytometric Analysis and Sorting]
Incubate at  for .
4 °C
?. [3.2.2 Fluorescent Staining for Flow Cytometric Analysis and Sorting]
At the same time, create single color controls for compensation by adding 1 drop of compensation beads to  and incubate with  for  at .
[cell resuspension buffer]
[one antibody]
4 °C
?. [3.2.2 Fluorescent Staining for Flow Cytometric Analysis and Sorting]
Wash samples and compensation beads with  and spin at .
[stain buffer]
Centrifuge: 400 34, 5 min
?. [3.2.2 Fluorescent Staining for Flow Cytometric Analysis and Sorting]
Resuspend the beads in  and filter into a FACS tube to ensure a single cell suspension ready for analysis and sorting on the BD FACS Aria.
[stain buffer]
?. [3.2.2 Fluorescent Staining for Flow Cytometric Analysis and Sorting]
To allow accurate gating of populations, create an FMO (Fluorescence Minus One) control for each antibody used. For example, the PE FMO control will contain every antibody used except the PE antibody.
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Set up the BD FACS Aria with a 70 μm nozzle and the pressure setting of medium drop drive frequency 60,000 with a “Purity” sort setting (e.g., 16-32-0; see Note 17).
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Run BD CS&T beads to confirm Aria laser performance.
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Run BD Accudrop beads to set up the drop delay and to align the 96-well plate.
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Ensure SSC-W, SSC-H, FSC-W and FSC-H are enabled to allow doublet discrimination.
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Add  to each well of a 96-well plate.
[5% BSA-supplemented PBS]
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Perform compensation using the single stained controls.
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Take the third of the original sample that was not passed through the autoMACS Pro and spin at  to pellet.
Centrifuge: 400 34
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Resuspend in —this is the “mixed” population of cells and does not requiring any specific sorting but requires the correct number of live cells to be counted into the 96-well plate.
[2% BSA-supplemented PBS]
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Gate the live, single cells on the basis of FSC/SSC. Sort 30,000 cells from within this gate into the appropriate well of a 96-well plate.
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Run the CD11b+ population through the Aria and gate on live single cells and then on F4/80+ cells to sort 30,000 macrophages (Fig. 4a).
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Run the CD11b− population through the Aria and gate on cells that are live/CD45−/CD326+ to sort 30,000 epithelial cells (Fig. 4b).
?. [Sorting of Mixed Cell Populations, Macrophages, Epithelial Cells, and Nonepithelial Cells into 96-Well 3.2.3 Plates for Downstream Assays]
Continue to run the CD11b− population, gating on cells that are live/CD45−/CD326− to sort 60,000 nonepithelial cells (Fig. 4a) (see Notes 18–20; Figs. 3 and 4).