The overall goals of these assays are to quantify monocyte chemotaxis in vitro in real time and to track lymphocyte migration in vivo. The in vitro method can help answer key questions in the field of immunology, such as what are the effects of serum factors on immune cell chemotaxis. The main advantage of the in vitro technique is that it permits quantitative and multiplex measurements in real time.
The in vivo method can help answer key questions in the field of immunology about how specific cell types migrate within a recipient animal. The in vivo technique is particularly useful for the quantitative assessment of the migration of multiple cell types within the same animal. Before beginning the experiment, label THP-1 cells with a fluorescent dye in a 35 millimeter cell culture dish in complete medium at 37 degrees Celsius for 30 minutes.
While the cells are incubating, add 750 microliters of HBSS buffered with 25 millimolar HEPES and 0.1%BSA to the background and positive control wells of a 24 well plate. Add the appropriate concentration of MCP-1 in water supplemented with BSA to the experimental and positive control wells. And place porous inserts into each well, taking care that the tabs of the inserts align with the notches of the plate.
It is critical to remove any bubbles from the bottom wells after placing the inserts, as bubbles will interfere with fluorescence measurements. At the end of the incubation, transfer the labeled cells into a 15 milliliter conical tube for their collection by centrifugation. And visually inspect the resulting pellet to confirm the uptake of the dye.
Next, carefully vacuum aspirate the supernatant and wash the cells in serum-free RPMI-1640 medium supplemented with 25 millimolar HEPES under the same centrifuge conditions. Resuspend the pellet in fresh serum-free medium with HEPES for counting. And adjust the volume to a final concentration of one to 1.5 times 10 to the six cells per milliliter.
Add 250 microliters of cells to the upper chamber of each well and place the plate into a 37 degrees Celsius plate reader. Then acquire fluorescence readings of the bottom chambers at one to three minute intervals at the appropriate fluorescence excitation and emission wavelengths for the dye. To prepare donor cells for their adoptive transfer, first, harvest the whole spleen from an anesthetized male eight to 12 week old C57 black six mouse into a 40 micrometer nylon mesh filter in a cell culture dish containing complete medium on ice.
Using the end of a syringe plunger, gently macerate the spleen through the mesh. When a single cell suspension has been obtained, transfer the cells into a 15 milliliter conical tube and add four volumes of ammonium chloride potassium lysis buffer to deplete the red blood cells. After five minutes at room temperature, collect the cells by centrifugation and resuspend the pellet in complete medium at a two to five times 10 to the seventh cells per milliliter concentration.
Strain the cells through a new 40 micrometer nylon mesh to remove the cell debris. And label one times 10 to the seven cells per recipient mouse with a live cell compatible fluorescent dye that will be retained upon subsequent fixation for 30 minutes at 37 degrees Celsius. At the end of the incubation, wash the cells in four volumes of HBSS supplemented with HEPES.
And resuspend the pellet in fresh HBSS plus HEPES to a one times 10 to the eighth cells per milliliter concentration. Next, briefly vortex the donor cells and load them into a one milliliter syringe equipped with a 27 gauge 0.5 inch needle. Place the anesthetized eight to 12 week old C57 black six recipient mice in the prone position and apply mild caudal pressure to the skin dorsal to the eye of the first animal, causing the eyeball to protrude slightly.
Direct the needle medially for the retro-orbital injection of 100 microliters of donor cells. Then, gradually withdraw the needle and place the mouse in an individual recovery cage with monitoring until full recovery. It is critical that the animals are appropriately anesthetized and that the injection procedure has been well practiced to ensure consistent cell delivery between the animals and across experiments.
Up to 24 hours after the adoptive transfer, harvest the spleens into one milliliter of complete medium per recipient. And generate a red blood cell-free single cell suspension as just demonstrated. After counting, adjust the cell suspension to a three times 10 to the seven cells per milliliter concentration in staining buffer.
And transfer 100 microliters of cells into individual wells of a 96 well round bottom plate. Stain the cells for the markers of interest according to the standard flow cytometric analysis protocols. After 20 minutes on ice protected from light, wash the cells in 100 microliters of staining buffer and resuspend the pellets in 200 microliters of fresh staining buffer for a second centrifugation.
Then resuspend the pellet in 125 microliters of fresh staining buffer and analyze the cell samples on a flow cytometer according to standard protocols. Automated fluorescent readings to track migration clearly demonstrate an induction of cell migration toward MCP-1 that is augmented in the presence of serum. Depending on the strength of the migratory stimulus, there is a lag of at least 15 minutes prior to an increase in fluorescent signal that may peak and gradually decline as the cells pass into solution into the lower chamber from the underside of the insert at a faster rate than that of the cells migrating from the upper chamber.
If more than one fluorescently labeled cell population is adoptively transferred, it is important to quantitate the relative proportion of cell populations within the input material to account for any variance in mixing the cell populations. For example, in this experiment, the ratio of green and orange fluorescent cells was 0.97 to one. The trafficking was quantified as the number of labeled cells recovered divided by the total number of cells recovered.
While there is considerable variance in the number of recovered cells between animals, for any given animal the ratio of green to orange fluorescent cells is typically approximately equal. Once mastered, the in vitro technique can be completed in four hours, and the in vivo technique can be completed in six hours if performed properly. While attempting this procedure, it's important to remember to avoid creating bubbles in the experimental chambers and to strive for consistency during the retro-orbital injection of the adoptively transferred cells.
After its development, this technique paved the way for researchers in the field of immunology to explore chemotaxis in vitro and in vivo. After watching this video, you should have a good understanding of how to conduct and monitor an in vitro chemotaxis assay in real time and isolate native lymphocytes for their adoptive transfer and for tracking their in vivo migration. Thanks for watching, and good luck with your experiments.
