This methodology allow us to quantify the chemotactic activity of blood monocyte in live animals and to study the effect of poor diets on monocyte function and their underlying mechanisms. Monocyte-derived macrophages can be isolated and analyzed using various approaches, including single cell, and omics techniques that provide a snapshot of the health of the blood monocyte site in mouse models. Today's procedure will be conducted by Dr.Young Joo Ahn an assistant professor in my lab, and Dr Luxi Wang, a postdoc in my lab.
Before beginning the procedure, clean the cell culture hood with disinfectant and completely thaw the growth factor-reduced basement membrane-derived solution on ice. Equip individual sterile one milliliter syringes with 26 gauge needles and place the needles on ice. Once the solution has thawed, wipe the top of the basement membrane-derived solution vial with an alcohol swab before loading 500 micro liters of solution supplemented with or without chemo attractant into each one milliliter syringe.
Then remove any air bubbles from the basement membrane-derived solution loaded syringe to ensure a uniform plug formation. After confirming a lack of response to toe pinch, slowly inject an entire volume of basement membrane-derived solution without MCP-1 added at approximately 100 micro liters per second subcutaneously into the right flank of the anesthetized mouse. And the entire 500 micro liters of basement membrane-derived solution supplemented with MCP-1 into the left flank of the animal.
Hold the syringe in place 20 to 30 seconds after the injection to prevent leakage of the solution and to allow a single, smooth gel plug to form before placing the mouse on a warming pad with monitoring until full recovery. Three days after the injection, remove the dorsal hair from a plug-injected animal and use forceps to grasp the skin around the lower thoracic and upper lumbar vertebra. Make a two millimeter long incision into the skin and cut along the midline of the back of the mouse from the cervical vertebrae down to one centimeter above the coddle vertebral junction.
Separate the skin from the muscle layer and pin down the skin on a polystyrene foam platform. Next, under a dissecting microscope, carefully grasp the fibrous capsule that contains the basement membrane-derived gel plug and use fine forceps to remove the fibrous capsule. Then use fine scissors to clean the plug.
Then transfer the cleaned basement membrane-derived gel plug into an appropriately labeled and weighed 1.5 milliliter micro centrifuge tube. Reweigh the tube to calculate the weight of the basement membrane-derived gel plug. For basement membrane-derived gel plug digestion, use clean fine scissors to mince the basement membrane-derived gel plug in each micro centrifuge tube and add 800 micro liters of displace to the plugs.
Vortex at the maximum speed for 10 seconds to disrupt the plugs and place the micro centrifuge tubes at 37 degrees Celsius for two hours in a thermo mixer at 1400 rotations per minute to completely dissolve the basement membrane-derived gel plugs. At the end of the incubation, sediment the plug fragments by centrifugation two times and resuspend the pellets in 300 microliters of PBS. Transfer 50 microliters of each cell suspension into new micro centrifuge tubes and label the cells in the new tubes with 0.5 microliters of calcine.
After a 10 minute incubation at 37 degrees Celsius in a carbon dioxide incubator, count the number of live green fluorescents positive and dead non-fluorescent cells on an automated cell counter. For a single cell Western blot analysis, add one milliliter of diluted single cell suspension to a rehydrated single cell Western blot chip in the bottom of a 10 centimeter petri dish. After five to 15 minutes, examine the chip under brightfield microscopy.
Approximately 15 to 20%of the micro wells should be occupied by a single cell and fewer than two percent of the wells should contain two or more cells. If the chip has been properly loaded, tilt the dish 45 degrees and wash the chip three times with suspension buffer to remove any uncaptured cells. After the last wash, carefully and load the chip into the electrophoresis cell of a single cell Western instrument, gel-side up, and cover the entire single cell Western blot chip with lysis running buffer.
Initiate the cell lysis and run the instrument according to the appropriate experiment parameters. At the end of the run, rinse the chip with two 10-minute washes with fresh washing buffer per wash at room temperature. After the second wash, add 80 microliters of the primary antibody solution of interest to the antibody probing chamber and lower the chip gel-side down so that the antibody solution wicks across the chip.
After two hours at room temperature, wash the chip three times in washing buffer and one time in water on a shaker. Incubate the chip with an appropriate secondary antibody for one hour at room temperature protected from light. At the end of the incubation, wash the chip three times with washing buffer and spin the chip on a slide spinner to remove any remaining washing buffer.
To strip the single cell Western blot chip, place a 15 milliliter tube rack in a 60 degrees Celsius water bath with the water just one centimeter above the rack. In a fume hood, add 40 milliliters of stripping buffer and 320 microliters of beta morcepta ethanol to a canister. Place the chip in a 10 centimeter petri dish inside the canister and seal the canister with Parafilm.
Then place the canister inside the tube rack in the water bath. After 90 minutes, carefully transfer the chip into a new petri dish and briefly wash the chip one time with wash buffer before adding 15 milliliters of fresh washing buffer to the petri dish for a 15 minute wash on the shaker. In this representative experiment, the cells recruited into each plug were counted at one, three, and five days after injection.
By subtracting the cell count in the vehicle-loaded plugs from the cell count in the MCP-1 loaded plugs, the number of cells specifically recruited in response to the chemo attractant were then calculated. An accelerated MCP-1 specific recruitment and accumulation of cells was observed over the five-day period, with rates increasing from 31, 000 cells per day after one day of injection to up to 136, 000 cells per day, five days after injection. Single cell Western blot analysis was then used to identify the different cell types recruited to the basement membrane-derived plugs, according to their expression of specific immune cell markers of interest.
For example, the percentage of monocytes within the MCP-1 loaded basement membrane-derived gel plugs was low at day one and peaked at day three before dropping again at day five, while the macrophage numbers increased steadily throughout the study period. For MCP-1 loaded plugs, the percentage of monocytes plus macrophages within the isolated cell population was highest at day three, indicating that after three days, the vast majority of cells recruited by MCP-1 dependent chemotaxis were monocytes and macrophages. Even though the total number of monocytes plus macrophages is typically higher at day five compared to day three, the cell count at day three more accurately reflects peripheral blood of monocyte chemotaxis and recruitment, and therefore day three is recommended for basement membrane-direct plug analyses.
Successful acquisition of single cells for downstream analysis depends on the accuracy of the injection and the complete removal of the plugs. Flow cytometry, single cell Western blotting, bulk, or single cell RNA sequencing and other omics procedures can be used to assess the characteristic and phenotypes of the recruited monocyte and monocyte-derived macrophages.
