Jungblut, M., Oeltze, K., Zehnter, I., Hasselmann, D., Bosio, A. Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator. J. Vis. Exp. (29), e1266, doi:10.3791/1266 (2009).
To work under sterile conditions, it is recommended to perform all steps in a laminar flow hood.
2. Dissociating the lung tissue
Part 4: Representative Results:
Figure 1. Lung dissociation with the gentleMACS™ Dissociator resulted in 92% viable cells. Dead cells were fluorescently stained with propidium iodide (PI) ( right dot plot; left dot plot: no PI staining).
Figure 2. Dissociation of mouse lung tissue using the gentleMACS Dissociator results in a high percentage of viable leukocytes and endothelial cells. The derived single-cell suspensions were stained with CD45-PE and CD146-FITC or CD31-FITC and analyzed by flow cytometry.
Figure 3. Single-cell suspension derived from mouse lung tissue was stained with CD11c-FITC and Anti-mPDCA-1-APC to detect mouse CD11clow m-PDCA-1+ plasmacytoid DCs as well as CD11chigh cells.
Figure 4. Enrichment of dendritic cells using CD11c MicroBeads, a MiniMACS Separator and two MS Columns.
In this video, we introduce a new method for the dissociation of mouse lung tissue. We show that a combination of mechanical and enzymatic treatment of lung tissue yielded a high percentage of viable leukocytes and endothelial cells. Specifically, the mechanical disintegration of the tissue was achieved by the gentleMACS Dissociator. The gentleMACS C Tubes include a rotor - stator system, which dissociates tissue in a gentle way. The procedure is controlled by the program settings of the instrument. The settings were optimized to achieve high yield and viability of cells. The derived single-cell suspensions were stained with CD45-PE and CD146-FITC or CD31-FITC and analyzed by flow cytometry to detect leukocytes and endothelial cells. Dendritic cells in the single-cell suspension were stained with CD11c-FITC and Anti-mPDCA-1-APC. Furthermore, dendritic cells from a mouse lung single-cell suspension were enriched using MACS Technology: dendritic cells were magnetically labeled using CD11c MicroBeads and separated using a MiniMACS Separator and MS Columns. 1,2 In general, the combination of our new dissociation protocol with magnetic cell sorting represents a standardized method to obtain specific cell types from lung tissue.
The authors are employees of Miltenyi Biotec GmbH, Germany.
|C Tubes||Miltenyi Biotec||Order no. 130-093-237||http://www.miltenyibiotec.com/en/PG_709_765_gentleMACS_C_Tubes.aspx|
|gentleMACS™ Starting Kit||Miltenyi Biotec||Order no. 130-093-235||http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx|
|MACSmix™ Tube Rotator||Miltenyi Biotec||Order no. 130-090-753||http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx|
|autoMACS™ Rinsing Solution||Miltenyi Biotec||Order no. 130-091-222||http://www.miltenyibiotec.com/en/PG_134_155_autoMACS_Rinsing_Solution.aspx|
|MACS® BSA Stock Solution||Miltenyi Biotec||Order no. 130-091-376||http://www.miltenyibiotec.com/en/PG_33_295_MACS_BSA_Stock_Solution.aspx|
|CD11c MicroBeads||Miltenyi Biotec||Order No. 130-052-001||http://www.miltenyibiotec.com/en/PG_93_132_CD11c_MicroBeads.aspx|
|CD11c-FITC, CD11c-PE||Miltenyi Biotec||Order no. 130-092-026130-091-830||http://www.miltenyibiotec.com/en/PG_599_329_CD11c.aspx|
|Anti-mPDCA-1-APC||Miltenyi Biotec||Order no. 130-091-963||http://www.miltenyibiotec.com/en/PG_599_325_Anti_mPDCA_1.aspx|
|CD146-FITC||Miltenyi Biotec||Order no. 130-092-026||http://www.miltenyibiotec.com/en/PG_599_417_CD146_LSEC_.aspx|
|CD45-PE||Miltenyi Biotec||Order no. 130-091-610||http://www.miltenyibiotec.com/en/PG_599_331_CD45.aspx|
|Propidium Iodide Solution||Miltenyi Biotec||Order no. 130-093-233||http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx|
|Art 1000 Reach Pipet Tips||Molecular BioProducts||Order no. 2079||http://www.mbpinc.com/ASP/products.aspx?query_TipID=61