High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The overall goal of the following experiment is to quantify the changes in the phagocytic process or act in polymerization following the addition of agonists or antagonists. This is achieved by adding fluorescently labeled particles to the phagocytic cells and allowing sufficient time for phagocytosis to take place. As a second step to stop phagocytosis cells are treated with triam blue to extinguish fluorescence of non internalized particles washed and later fixed with paraldehyde.

Next is staining and quantification. In order to facilitate fluorescent detection of cells and particles, cells sustained with DPI or actin, the results show the effect of treatment on phagocytosis and actin polymerization as quantified through a ratio green red fluorescence to blue based on fluoro metric analysis. The main advantage of the fluoro metric technique over existing methods is that it is a high throughput cost-effective method for assessment of phagocytosis.

It allows for minimal sample manipulation and quick quantification of fluorescent intensity per cell. This method can help answer key questions in the field of immunology and cell biology, such as how effective and how fast this pathogen clearance is. Action polymerization changing during phagocytosis and how is phagocytosis or axon polymerization modulated by agonists or antagonists.

To begin the procedure plate, the macrophages from the Murine J 7 74 cell line in a 96 well plate with a clear or black bottom incubate the adherence cells at 37 degrees Celsius for one to two hours to allow adion to the plate. Then add the desired agonists or antagonists or vehicle control to the cells at two x concentration in 50 microliters of PBS or supplemented media and incubate for a desired amount of time. In this step, ensure that the fluorescent particles and labels are protected from light at all times during processing, washing, and incubation.

In order to prevent photobleaching to use the particles in suspension such as fluorescently labeled dextra beads orex the stock solution for one minute. Next, add an equal amount of opsonizing reagent to the fluorescent particles and vigorously vortex for another minute. Subsequently, incubate the mixture at 37 degrees Celsius for one hour following incubation centrifuge the particles were five minutes.

At 500 times G.Remove the S supernatant containing excess opsonizing reagent. After that, wash the particles with 100 microliters of PBS. Vortex the sample to resuspend the pellet and centrifuge it for five minutes of 500 to times G.Repeat these steps two more times to assure proper optimization and removal of unbound antibody.

To prepare the working solution of ionized particles, re suspend the particles in five milliliters of media. Then store it away from light until further usage. Now place the working solution of particles in a sterile reagent reservoir.

Add 50 microliters of opsonize particles to the cells prepared earlier to a final concentration of 15 nanograms per milliliter, and allow the phagocytosis to occur at standard cell culture conditions. For the continuous phagocytosis method, allow 30 minutes to one hour for the fluorescent particles to reach the bottom of the well and to get in touch with the cells. If conducting a time course experiment, add bacteria at different time intervals and stop the entire plate of reactions at the same time.

To enhance the speed of processing for adherence cells, remove the sennet and add 50 microliters of diluted triam blue with PBS in order to extinguish fluorescence of non internalized bacteria following trian incubation. Wash the plate two times with PBS to remove any residual trian. Next, fix the cells with 50 microliters of 4%paraldehyde and incubate for 15 minutes at room temperature.

Then wash the plate with PBS. Remove all the liquid and add 50 microliters of DPI After five minutes of staining, wash the plate twice with PBS. Subsequently, add 50 microliters of PBS to each well and record phagocytosis following paraldehyde fixation.

Wash the plate twice with 100 microliters of PBS per well for each wash. Then add 50 microliters of 0.1%Triton X 100 in PBS to the cells for permeation and incubate them for three to five minutes at room temperature. Next, wash the 0.1%Triton X away with PBS twice.

Subsequently block the cells with 1%BSA for 20 minutes to avoid non-specific binding of the label. Now prepare the working solution of Rod domine stain by adding 100 microliters of methanol stock solution in five milliliters of PBS. Following blocking, remove the BSA solution immediately.

Add 50 microliters of rumine foid and working solution to each well and incubate the cells in the dark for 20 minutes of room temperature. To quantify phagocytosis, place the plate in a fluorescent plate reader Record green fluorescence using an excitation filter at a wavelength of 488 nanometers and an emission filter at a wavelength of 518 nanometers and blue fluorescence using an excitation filter at a wavelength of 355 nanometers and an emission filter at a wavelength of 460 nanometers. To quantify actin polymerization using a fluorescent plate reader record red fluorescence via an excitation filter at a wavelength of 584 nanometers and an emission filter at a wavelength of 620 nanometers and blue fluorescence.

Fire and excitation Filter at a wavelength of 355 nanometers and an emission filter at a wavelength of 460 nanometers. Next, divide the total RFU of green or red fluorescence by the blue fluorescence readout depending on the particle label or phagocytic versus actin quantification due to high plate. To plate variance, use relative indexes to best illustrate the trends observed and to facilitate more reliable statistical analysis.

This image illustrates the changes in actin polymerization following the addition of inhibitor. These are the pseudopodia formation and here are the changes in membrane ruffling. In this image, the cells were allowed to internalize fitzy conjugated X beads at a 20 to one bead to cell ratio for 60 minutes.

Then the cells will wash with trian and PBS fixed with paraldehyde, permeable with acetone and stain for F actin Using rod domine, F loin and dpi, continuous phagocytosis of opsonize and unoxidized particles is quite comparable. Where J 7 74 cells are internalizing dextra beads. Interestingly, actin polymerization following phagocytosis increases slowing down at later time points while the actin polymerization following internalization of unop particles does not increase neither of the phagocytic methods using dextra particles results in changes in cell viability Once mastered, this technique can be done in one to two hours with multiple plates at a time if it is performed properly.

After watching this video, you should have a good understanding of how to utilize Fluor metric analysis as an efficient, cost-effective high throughput method of analyzing phagocytosis and other cellular processes.