Isolation of Mouse Lung Dendritic Cells

1. Lung perfusion and single cell suspension

1. Euthanize the mouse with an intraperitoneal injection of ketamine/xylazine anesthetic mixture (in mg/mouse ketamine 1.8, xylazine 0.19) and exsanguination via the femoral vein
2. Expose the thoracic cavity by cutting and gently pulling back the outer skin of the peritoneum. Proceed to open the diaphragm by cutting the rib cage to expose both the heart and lungs.
3. Perfuse gently the lungs using a 5 ml syringe filled with EDTA-HBSS (1mM EDTA in calcium/Magnesium-free HBSS). Connect a 25 G needle and insert needle into the right ventricle. To prevent leakage during injection, secure the myocardiac tissue around the needle by using forceps. Gently inject solution while maintaining a constant pressure. Accurate perfusion will result in lung inflation and a color change to pink/white.
4. Remove draining lymph nodes to discard any potential contamination from this tissue.
5. Collect lung tissue, transfer to a petri dish on ice, and chop it to small pieces using a razor blade.
6. Transfer grounded tissue into a gentleMACS C tube containing 5 ml/lung of collagenase digestion solution (collagenase type 1A 0.5 mg/ml plus type IV bovine pancreatic DNase 20 μg/ml in HBSS containing 5% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin).
7. Use the gentleMACS dissociator to obtain a single cell suspension. Choose program: m_lung_01. Incubate sample at 37 °C for 30 minutes. Shake tube every 5 minutes to resuspend tissue fragments. Proceed with program m_lung_02.
8. Transfer sample to a 15 ml conical tube and centrifuge for 10 minutes at 335 x g at 4 °C. After this step, keep all reagents and centrifugations at 4 °C to prevent reduction in cell viability.
9. Discard supernatant and lyse remaining red blood cells by adding 2 ml of ACK lysing buffer for 1 minute at room temperature. Wash cells with 13 ml of cold PBS/0.5% BSA and centrifuge for 10 minutes at 335 x g at 4°C.
10. Discard supernatant, resuspend total cells in 5 ml of cold PBS/0.5%BSA and pass thorough a 100 μm nylon mesh.
11. Determine the total cell number by using trypan blue exclusion dye.

2. Magnetic isolation and CD11c⁺ cells enrichment

1. Centrifuge single cell suspension for 10 minutes at 200 x g at 4 °C and discard supernatant.
2. Resuspend cell pellet in 400 μl of separation buffer (PBS, 0.5% BSA, and 2 mM EDTA) per 10⁸ total cells. Block Fc-mediated unspecific antibody binding by using anti-CD16/CD32 antibody (0.5μg/ 10⁶ cells) for 30 minutes at 4 °C.
3. Wash cells with 10 ml of cold separation buffer and centrifuge for 10 minutes at 200 x g at 4 °C. Resuspend cells in 400 μl of separation buffer.
4. Add 100 μl of CD11c MicroBeads per 10⁸ total cells. Mix well and incubate for 15 minutes in the refrigerator (2-8 °C).
5. Wash cells with 10 ml of cold separation buffer and centrifuge for 10 minutes at 200 x g at 4 °C and discard supernatant.
6. Resuspend the cell pellet in 3 ml of cold separation buffer.
7. Perform magnetic separation using the autoMACS Pro^TM Separator by selecting the program posseld. Collect the positive fraction (0.5 ml).

3. Conventional dendritic cell (cDC) isolation

1. Incubate enriched CD11c positive cell suspension with anti-CD11c PE-Cy7 and anti-I-A/I-E (MHC-II)-FITC antibodies for 30 minutes at 4 °C. Optimized concentration of antibodies is 0.5 μg/ 10⁶ of cells.
2. Wash cells with cold PBS/0.5% BSA and centrifuge for 10 minutes at 335 x g at 4 °C. Resuspend cells in 0.5 ml of PBS/0.5% BSA and pass them through a 40 μm nylon mesh.
3. Proceed to sort the cells by using the FACS Aria cell sorter in the purity mode. Add 100 μl of PBS/0.5% BSA to the collection tubes in order to prevent damage of purified cells. Keep your cells at a refrigerated temperature throughout the cell sorting process.

4. Alternate protocol for obtaining a single cell suspension and CD11c+ cells enrichment

1. In replacement of the gentleMACS dissociator, grounded lung tissue from step 1.5 can be transferred to a 15 ml conical tube containing 5 ml of collagenase solution per lung and incubate for 1 hour at 37 °C. Vortex cells every 15 minutes in order to resuspend tissue fragments. Disrupt the tissue by passing the sample 6-8 times in and out through a 3 ml syringe connected to a 20 G needle. Avoid making bubbles during the process, otherwise the cell viability will be compromised. Once single cell suspension is obtained, continue to step 1.8.
2. In replacement of the AutoMACSPro^TM separator, magnetic isolation for the enrichment of the CD11c cells, can also be performed by passing the cell suspension manually through two MACS columns. The selection of the appropriate columns will vary depending of the sample size.

5. Representative Results:

Lung cDC are identified as CD11c^hi/MHC-II^hi cell population. As shown in Fig. 1, cDC represent a smaller percentage of 1.4% when compared to some other tissues such as spleen (4.8%). However, in contrast to spleen, lung CD11c positive cells express different amounts of MHC-II, including a cell population different than that of cDC. After the single-cell preparation, the cell yield of total lung cells was about 3.0 to 3.8 x 10⁷ cells/lung with a viability of 60-70 % from which a little over 1% represented cDC subset. This percentage was increased after the CD11c magnetic isolation where the total lung cDC was incremented about 10 times (>16%) from the original preparation (Fig. 2A). However, CD11c cells expressing low amounts of MHC-II (CD11c^hi/MHC-II^lo, macrophages) represented a high contaminating cell population (>70%) which was mixed with the lung cDC subset. As shown in Fig. 2B, those cells were eliminated after the cell sorting step when the cDC purity reached >96%. The usual yield of cDC after the whole procedure was 5 x 10⁴ cells/lung. Microphotographs show morphological characteristics of cDC as rounded cells with the typical dendrites (upper panel). On the other hand, CD11c^hi/MHC-II^lo represented a morphologically different subset that shows cellular protuberances which are typical of macrophages (MΦ, lower panel) ¹².

Figure 1. Differential DC frequency in mouse lung and spleen. Lung and spleen tissue from C57BL/6 mice were collected and treated with collagenase. Following collagenase digestion, cells were stained with anti-mouse CD11c-PE-Cy7 and anti-mouse I-A/I-E-FITC. Representative flow cytometry plots show percentages of cDC (CD11c^hi/MHC-II^hi) in lung and spleen.

Figure 2. Isolation of cDC from mouse lung. Mouse lung single cell suspension was labeled with anti-CD11c microbeads and passed through an automatic cell separator. A) A representative plot shows the percentages of cDC (CD11c^hi/MHC-II^hi) and Macrophages (MΦ, CD11c^hi/MHC-II^lo) populations after CD11c enrichment. Further staining of the enriched fraction followed using anti-mouse CD11c-PE-Cy7 and anti-mouse I-A/I-E-FITC. Double positive cells were sorted and analyzed by flow cytometry. To identify their morphology, cells were cytospun and stained with a modified Wright-Giemsa staining. B) Representative plots show percentages of cDC and MΦ after cell sorting. Microphotographs show a representative image of lung cDC and lung MΦ. Scale bar = 20 μm.