Exploring the Sequential Cellular Events of Phagocytosis Triggered by Godanti Bhasma in Mammalian Cells

Phagocytosis, where a phagocyte ingest and breakdown foreign particles. It's crucial for immune defense. Improved user-friendly methods are needed to efficiently monitor and evaluate this complex cellular process.

Capturing real-time degradation events and avoiding artifacts from photo bleaching remain key challenges in accurately studying phagocytosis and intracellular trafficking. Unlike fluorescent or synthetic particle, our protocol uses Godanti Bhasma, a label-free particle enabling direct visualization of phagocytosis stages without photo bleaching, optimization or complex preparation. These findings provide a reproducible model to dissect phagocytic mechanisms, offering new tools to study vesicular dynamics, pathogen dysfunction, and potential therapeutic intervention to immune and lysosomal disorders.

We aim to explore the molecular regulators of phagosome of maturation, investigate phagocytic dysfunction in disease models, and screen potential drugs that modulate vesicular dynamics and intracellular trafficking. To begin, weigh exactly 100 milligrams of Godanti Bhasma or GB powder. Suspend the powder in 10 milliliters of DMEM supplemented with 10%FBS and 1%penicillin streptomycin.

Vortex the suspension thoroughly to mix the contents and let the mixture stand for one minute to allow the larger particles to settle at the bottom. Using a pipette, transfer the upper five milliliters of the suspension into a sterile centrifuge tube. Label the five milliliters of transferred GB suspension as the stock solution for further cell culture work.

Vortex the suspension thoroughly again to ensure even dispersion of particles before adding it to the cells. Seed 3 10 to the fourth power 3T3-L1 cells into a 35 millimeter dish containing two milliliters of DMEM. Incubate the dish at 37 degrees Celsius in an atmosphere of 5%carbon dioxide until the cells reach 70%confluency.

Then mix 300 microliters of the GB stock suspension thoroughly into two milliliters of prewarmed fresh culture medium. Place the culture dish containing cells and GB suspension onto the stage incubator of the imaging microscope. Select the 10x objective on the microscope.

Turn on the bright field illumination using the Bright button. Then launch the DIGI ImagePlus software and select the appropriate camera. Click the Capture button and choose the time lapse option.

Set the interval time to five minutes and start recording by clicking the Finish button. Capture bright field images at five minute intervals continuously for 16 to 24 hours inside the microscope stage carbon dioxide incubator. Use ImageJ software to compile all captured images into a time-lapse video.

This enables identification of the phagocytosis stages from particle internalization to their complete degradation. After 24 hours of cell growth in 96 well plates, mix the GB particle stock suspension thoroughly. Add 30 microliters of the suspension to each well.

Use wells without GB particles as negative controls. Incubate the plates at 37 degrees Celsius in 5%carbon dioxide for 24 hours to induce vacuole formation. To prepare neutral red solution, dissolve neutral red to make a 0.5 milligrams per milliliter solution in serum free DMEM.

Filter the solution through a 0.2 micrometer filter. For imaging, remove the media from 96 well plates. After adding 60 microliters of the filter dye to each well, incubate the plate at 37 degrees Celsius for 15 minutes.

Wash the wells three times with PBS to remove excess dye. Then proceed with microscopic imaging. Examine the stained vacuoles under a microscope using the 20x objective to assess the size, morphology and number of vacuoles.

To prepare bafilomycin A1 stock solution, dissolve 100 micrograms of the compound in 50 microliters of dimethyl sulfoxide. Then dilute it with 950 microliters of DMEM. For the working solution, dilute one microliter of the stock in 1.6 milliliters of DMEM to obtain a final concentration of 0.1 nanomolar.

Mix the working solution thoroughly and add 100 microliters to the wells containing cells in the 96 well plates. Then incubate the cells in each well with 30 microliters of GB particle suspension to study inhibition of vacuole and phagosome formation. To inhibit phagosome acidification, add 100 microliters of the bafilomycin A1 working solution to wells with preformed vacuoles previously treated with GB.Incubate all wells at 37 degrees Celsius in 5%carbon dioxide for 24 hours.

Add 60 microliters of neutral red dye to each well. Incubate for 15 minutes at 37 degrees Celsius and wash the wells three times with PBS to remove excess dye, then analyze vacuole formation and acidification under the microscope. Treat the cultured cells with 50 microliters of GB suspension and incubate them overnight at 37 degrees Celsius in 5%carbon dioxide under standard experimental conditions.

Following treatment, add acridine orange solution at one milligram per milliliter concentration to the cells and incubate for 15 minutes at 37 degrees Celsius to stain vacuoles and acidic vesicular organelles. Next, wash the cells three times with sterile PBS to remove any unbound dye and reduce background fluorescence. Finally, analyze the stained slides immediately using a fluorescence microscope fitted with a 20x objective and a blue excitation filter range of 450 to 492 nanometers.

Assess cellular morphology and acridine orange uptake to understand vacuolar dynamics. After 30 minutes of GB treatment, particles were observed attached to the surface of three 3T3-L1 cells. A cup like membrane extension appeared around the particle indicating the initiation of particle engulfment.

At 12 hours post-treatment, large membrane-bound vacuoles containing GB particles were observed inside the cells. Time-lapse imaging demonstrated the stepwise internalization and vacuole formation in a single live cell over 4.75 hours. GB particles within vacuoles progressively degraded over time and the cell regained normal morphology as indicated by vacuolar turnover.

Flow cytometry analysis showed that internalized Godanti Bhasma particles were completely degraded within 24 hours with no particles remaining in the culture medium. Neutral red staining revealed significantly higher vacuole acidification in GB treated cells of all three cell lines compared to controls which displayed only weak lysosomal staining. Acridine orange staining showed orange red fluorescence in vacuoles of GB 3T3-L1 cells indicating acidification, while untreated cells showed only green fluorescence.

Co-treatment with bafilomycin inhibited vacuole formation in both 3T3-L1 and HeLa cells as evidenced by the absence of vacuoles compared to their respective controls. After treatment with bafilomycin, neutral red uptake was effectively blocked disrupting vacuole acidification and maturation in cells.