An In Vitro Method to Identify Innate and Adaptive Immune Cells Residing in Murine Lungs

Transfer the lung to a Petri dish. Mince it using two fine scalpels, and then place it into a 50-milliliter conical tube. Add 5 milliliters of digestion buffer to wash the plate. Secure the lid of the tube, and digest the lung for 30 minutes on an orbital shaker at a speed of 150 rotations per minute at 37 degrees Celsius. Stop the reaction by adding 10 milliliters of cold BSA buffer.

After digestion, mix and dissolve the lung pieces using an 18-gauge needle. Place a 70-micrometer filter strainer on top of a new 50-milliliter conical tube, and transfer the digested lung mixture into the strainer.

Use the rubber side of a 10-milliliter syringe plunger to smash the remaining lung pieces on the filter, and wash it with BSA buffer. Centrifuge the single-cell suspension at 350 g for 8 minutes at 7 degrees Celsius. Discard the supernatant, and resuspend the cells in 1 milliliter of ACK lysis buffer. Mix the suspension using a 1-milliliter pipette, and incubate it for 90 seconds at room temperature.

Add 10 milliliters of cold BSA buffer to the reaction mixture, and centrifuge it at 350 g for 7 minutes at 4 degrees Celsius. Discard the supernatant. Resuspend the pellet in staining buffer, and count the cells using a hemocytometer. Resuspend the cells at a concentration of 5 million cells per milliliter for surface staining.

Transfer 1 million cells in 200 microliters per well into a 96-well plate, and centrifuge the plate at 350 g for 7 minutes at 4 degrees Celsius. Prepare a flow cytometry block solution by diluting anti-16/32 antibody in staining buffer.

Resuspend the cells in 50 microliters of the flow cytometry block solution. Incubate the suspension for 15 to 20 minutes at 4 degrees Celsius, or on ice. Then, add 150 microliters of staining buffer to the plate, and centrifuge it at 350 g for 5 minutes at 4 degrees Celsius.

Prepare surface antibody solution by diluting the surface antibodies in staining buffer. Resuspend the cells in 50 microliters of the surface antibody solution, and incubate the plate at 4 degrees Celsius in the dark. Then, wash the cells twice with staining buffer.

Prepare the fixation and permeabilization buffer by mixing three parts fixation and permeabilization diluent, and one part staining buffer. Resuspend the cells in 50 microliters of the preprepared buffer per well of the 96-well plate, and incubate them for 20 to 25 minutes at 4 degrees Celsius in the dark.

Dilute the permeabilization buffer with purified deionized water to prepare one times permeabilization buffer, and use it to wash the cells. Prepare an intracellular antibody solution by diluting with 1 milliliter of permeabilization buffer. Resuspend the cells in 50 microliters of the diluted intracellular antibody solution per well of the 96-well plate, and incubate for 40 minutes at 4 degrees Celsius in the dark.

Wash the cells with permeabilization buffer, then with staining buffer. After the final wash, resuspend the cells in 200 microliters of staining buffer. Acquire a minimum of 1.5 million cells per sample on the flow cytometer.