Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

In this video protocol, you will see how to dissociate adult mouse lung tissue using gentle max following dissociation of lung tissue. In the presence of collagenase d and DNAS one cell suspensions are filtered using a 70 micron cell strainer centrifuge, and suspended for further applications. Hi, I'm Melanie Mlu from Mil Biotech in Germany.

Today I'll show you a reproducible and automated procedure for the preparation of a single cell suspension out of mouse lung tissue by using the GenX dissociation. We use this procedure in our lab to prepare single cell suspensions for the preparation of leukocytes or endothelial cells. Okay, let's get started.

Before we begin the protocol, a few standard solutions must be prepared and at the ready. First, we will need Hebes buffer. Second, a collagenase D solution containing 100 milligrams per milliliter enzyme and hebes buffer.

Third, a dase one solution containing 20, 000 units per milliliter dase one. Lastly, two common buffering solutions. A typical PBS buffer at pH 7.2 and A PEB buffer derived from AutoMax rinsing solution and BSA stock solution.

We begin our protocol with a dissected lung derived from a six to seven week old female biopsy mouse. Make sure that the lung is free of adjoining organs such as the heart and thymus, efferent and afferent blood vessels, trachea and connective tissues. Rinse the tissue in a Petri dish containing PBS to remove erythrocytes.

Typically, a mouse lung at this age weighs between 110 and 150 milligrams. If you plan to associate lungs from older animals or other mouse strains, weigh the tissue and buffer. A maximum of 450 milligram tissue can be dissociated per one tube.

Transfer the lung tissue to a gentle max C tube loaded with 4.9 milliliters of heis buffer. Add 100 microliters of collagenase D solution to a final concentration of two milligrams per milliliter to the lung tissue and buffer. Now 10 microliters dase.

One solution for more than 150 milligram tissue. Add 20 microliter dase one solution. Make sure the C tube is tightly closed and place it upside down onto the gentle max dis.

Make sure that the tissue is located between the rotor and the stator. Select the lung program and run it by pressing the start button when the program ends. Remove the C tube from the instrument and incubate the sample for 30 minutes.

In a 37 degree Celsius incubator during the incubation, the tube should be rotated using the max mix tube rotator or manually turned every five minutes. After the incubation, return the C tube to the gentle max Associator and run the second lung program While the program is running, prepare a 50 milliliter tube for collecting filtered cells by replacing the cap with a 70 micrometer mesh cell strainer. Once the dissociated program ends, depending on the distribution of sample material in the tube, you may want to centrifuge the tube briefly to concentrate the sample material.

The dissociated cells can be removed from the tube by pipetting through the septum sealed cap of the C tube. Using a suitable 1000 microliter pipette tip, the cell suspension is applied to the cell strainer to remove larger particles or aggregates. Once the solution has drained, wash the cell strainer by adding an additional five milliliters of room temperature and hees buffer to pellet the filtered cells.

Centrifuge the 50 milliliter tube at 300 GS for 10 minutes. Now aspirate the supernatant and resuspend the cell pellet in your desired volume of room temperature. PEB buffer.

Do not decant the supernatant. You can now proceed to your downstream application. Dissociation of mouse lung tissue using this procedure will yield a high percentage of viable leukocytes and endothelial cells.

The derived single cell suspensions were stained with CD 45 PE as CD 1 46 FITC, or CD 31 FITC, and analyzed by flow cytometry. This plot shows the enrichment of dendritic cells using CD 11 C microbeads, a minimax separator, and two MS columns.Okay. I've just shown you how to prepare single cell suspensions out of mouse lung tissue in a standardized way by using the gentle mix dissociation and C tubes.

This method allows the efficient and gen tissue dissociation. When doing this procedure, it's important to properly dissect the lung and process the tissue immediately after dissection. The prepared single cell suspensions can then be used for further experiments.

For example, the magnetic labeling of the cells, and then the separation of leukocytes or endothelial cells. Okay, that's it. Thank you for watching and good luck with your experiments.