Name: Exploring the Sequential Cellular Events of Phagocytosis Triggered by Godanti Bhasma in Mammalian Cells (Video) | JoVE Journal
Uploaded: 2025-07-11T15:50:00.000+00:00
Duration: 10 min 10 s
Description: Explore phagocytosis mechanisms using Godanti Bhasma in 3T3-L1 mammalian cells via time-lapse microscopy. This label-free method enables direct visualization of particle uptake, vacuole dynamics, and degradation without photobleaching artifacts.

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5 minutes

This research was supported by the Science &#38; Engineering Research Board (SERB) Core Research Grant (CRG/2022/009045), Department of Science and Technology, Government of India.

1. Cell culture and seeding
<ol>
	<li>Prepare complete DMEM medium supplemented with 10% heat-inactivated FBS and antibiotics (100 &#181;g/mL penicillin, 100 &#181;g/mL streptomycin). Sterilize all reagents and equipment before use.</li>
	<li>Culture the 3T3-L1, HeLa, and RAW 264.7 cells in 25 cm2 flasks and 35 mm culture dishes using DMEM. Maintain the cultures at 37 &#176;C in a humidified incubator with 5% CO2 and replace the medium every 48 h.</li>
	<li>For subculturing, wash the cells 2x with PBS, then detach adherent cells using 500 &#181;L of Trypsin-EDTA and incubate for 5 min. Add 1 mL of DMEM to neutralize the Trypsin-EDTA solution, pipette to create a single-cell suspension, and adjust the volume to 10 mL with fresh DMEM. Centrifuge at 300 x g for 5 min, resuspend the pellet in 1 mL of fresh medium.</li>
	<li>Mix 10 &#181;L of cell suspension with 0.4% trypan blue (1:1), count viable cells using a hemocytometer, calculate density as Cells/mL = Avg. count x 104 x 2.</li>
	<li>For 96-well plates: Dispense 100 &#181;L of the cell suspension (containing 10,000 cells) into each well. For cavity slides, seed 50 &#181;L of the cell suspension (containing 5000 cells) into two cavities, leaving the middle cavity empty. Add 50 &#181;L of DMEM to each of the seeded cavities.</li>
	<li>Incubate the plates and slides at 37 &#176;C in a 5% CO2 for 24 h to allow cell adherence and stabilization. Inspect the cells under an inverted microscope at 20x magnification to assess morphology, confluency, and contamination before proceeding with the experiments.</li>
</ol>
2. Preparation of GB stock solution/cocktail for cell culture
<ol>
	<li>Procure GB and weigh 100 mg of the powder precisely. Suspend the powder in 10 mL of DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.</li>
	<li>Vortex the suspension and let it stand for 1 min to allow larger particles to settle. Then, transfer the upper 5 mL of the suspension into a sterile centrifuge tube.</li>
	<li>Designate this 5 mL prepared GB suspension as the stock solution for subsequent cell culture experiments. Vortex the suspension thoroughly to ensure even dispersion of the particles before adding it to the cells.</li>
</ol>
3. Flow cytometry analysis
<ol>
	<li>Harvest RAW 264.7, HeLa, or 3T3-L1 cells by trypsinization, neutralize with complete DMEM, centrifuge at 300 x g for 5 min, resuspend in 1 mL of fresh medium, mix 10 &#181;L with 0.4% trypan blue (1:1), count viable cells using a hemocytometer, calculate density and adjust to 2 x 105 cells/mL.</li>
	<li>Seed 2 x 105 cells per well in a 12-well plate and culture for 24 h. Incubate at 37&#8239;&#176;C with 5% CO2.</li>
	<li>Treat the cells with GB suspension (300 &#181;L per well) for various durations: 30 min, 1 h, and 12 h. After treatment, wash the cells 5x with PBS to remove excess particles.</li>
	<li>Trypsinize the cells as in step 1.3 and wash them thoroughly with PBS. Load the prepared suspension into the sample port of the flow cytometer. Launch the software and select the appropriate acquisition settings. Choose Bright Field Mode for imaging.</li>
	<li>Start data acquisition by clicking Run in the software. Acquire data for at least 10,000 events as the system captures images of individual cells passing through the flow cell.</li>
	<li>Use area versus aspect ratio scatter plots to gate single intact cells. This helps exclude doublets and clusters. View the corresponding images in the image library. Collect individual cell images from the software image library to study the morphological changes induced by the treatment.</li>
	<li>For particle analysis, seed 2 x 105 cells per well in 12-well plates and culture for 24 h. Treat the cells with GB suspension (300 &#181;L per well) for different durations: 1 h, 7 h, 14 h, and 24 h.</li>
	<li>After treatment, gently shake the culture plate for 1 min and collect the culture media into 1.5 mL tubes. Wash the cells 5x with PBS to remove any extracellular particles.</li>
	<li>Add 500 &#181;L of RIPA buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% Triton X-100; 1% sodium deoxycholate) to completely lyse the cells.</li>
	<li>Run the culture medium and lysed cell samples containing particles separately through the flow cytometer. Load the samples into the sample port of the system. Launch the software and select the appropriate acquisition settings. Choose Bright Field Mode for particle analysis.</li>
	<li>Start data acquisition by clicking Run in the software. Acquire data for at least 20,000 events as the system captures images of individual particles passing through the flow cell.</li>
	<li>Use area versus aspect ratio scatter plots to gate all particles. Use the scatter plot tools to analyze particle count.</li>
</ol>
4. Time-lapse microscopy
<ol>
	<li>Culture 3T3-L1 cells (3.0 x 104) in a 35 mm dish with 2 mL of DMEM at 37&#8239;&#176;C in a 5% CO2 until they reach 70% confluency.</li>
	<li>Thoroughly mix 300 &#181;L of GB suspension into the pre-warmed fresh 2 mL culture medium and then place the culture dish with the cells and GB, on the imaging microscope stage.</li>
	<li>Select the 10x Objective. Turn on the brightfield illumination using the Bright button. Click the Movie button to enter time-lapse mode. Set the interval time to 5 min. Start recording by clicking the Rec button.</li>
	<li>Capture images at every 5 min intervals for 16 h to 24 h, ensuring continuous imaging under standard conditions in a CO2 incubator.</li>
	<li>Use ImageJ software to compile the captured images into a time-lapse video. This technique allows for the differentiation of various stages of phagocytosis, from internalization to complete degradation of particles.</li>
</ol>
5. Neutral red staining
<ol>
	<li>After 24 h of cell growth in 96-well plates and cavity slides, thoroughly mix the GB particle suspension stock and add 30 &#181;L of the suspension to each well or cavity.</li>
	<li>Incubate at 37 &#176;C with 5% CO2 for 24 h to induce vacuole formation. Use untreated wells or cavities as negative controls.</li>
	<li>For Neutral Red (NR) staining, prepare a 0.5 mg/mL NR solution in serum-free DMEM, and filter it through a 0.2 &#181;m filter.</li>
	<li>For imaging of vacuoles in 96-well plates and cavity slide: remove the media, add 60 &#181;L of NR dye, and incubate at 37 &#176;C for 15 min. Wash 3x with PBS to remove excess dye and then proceed with microscopic imaging.</li>
	<li>Examine the vacuoles under a microscope using the 20x objective, to assess size, morphology, and number of vacuoles.</li>
</ol>
6. Treatment with Bafilomycin A1 for vacuole formation and acidification inhibition
<ol>
	<li>Prepare a stock solution of Bafilomycin A1 (BFA1) at a concentration of 100&#8239;&#181;g/mL (160.5&#8239;nM) by dissolving 100&#8239;&#181;g of BFA in 50&#8239;&#181;L of DMSO, followed by dilution in 950&#8239;&#181;L of DMEM. Dilute 1 &#181;L of this stock in 1.6 mL of DMEM to obtain a working solution of 0.1 nM.</li>
	<li>Mix thoroughly and add 100 &#181;L of the working BFA solution to wells containing cells in the 96-well plates. Also, add 30 &#181;L of GB particle suspension to assess inhibition of vacuole and phagosome formation.</li>
	<li>To inhibit phagosome acidification, add 100 &#181;L of the working BFA solution to wells with pre-formed vacuoles (previously treated with GB particles). Incubate all wells at 37 &#176;C with 5% CO2 for 24 h.</li>
	<li>Add 60 &#181;L of Neutral Red (NR) dye to each well, incubate for an additional 15 min, and analyse vacuole formation and acidification.</li>
</ol>
7. Acridine orange (AO) staining
<ol>
	<li>Treat the cells cultured in cavity slides with 50 &#181;L of GB and incubate overnight under standard experimental conditions.</li>
	<li>After treatment, incubate the cells with acridine orange solution (1 mg/mL) for 15 min at 37 &#176;C to enable selective staining of vacuoles and acidic vesicular organelles.</li>
	<li>Wash the cells 3x with sterile phosphate-buffered saline (PBS) to remove unbound dye and minimize background fluorescence.</li>
	<li>Quickly analyze the slides with a fluorescence microscope equipped with a 20x objective and a blue excitation filter range of 450-492 nm, to assess cellular morphology and acridine orange uptake, which will provide insights into vacuolar dynamics.</li>
</ol>

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The authors declare no conflicts of interest.

Phagocytosis serves as a crucial immune defense mechanism, enabling the engulfment and subsequent elimination of particles, pathogens, and apoptotic cells. To understand the processes of phagosome formation and maturation, smart biocompatible particles combined with advanced imaging techniques are essential for studying dynamic events such as the vacuolar progression, acidification, and degradation of engulfed materials18,19. We employed GB, a biocompatible model...

Phagocytosis is an essential cellular process carried out by tissue-resident professional phagocytes, such as macrophages and dendritic cells, as well as non-professional phagocytes, such as epithelial cells and fibroblasts, which engulf and degrade foreign particles. These cells internalize and eliminate foreign particles, pathogens, or cellular debris, contributing significantly to immune defense and tissue homeostasis1. This process begins with the recognition of pathogens, debris, or particles through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), which bind to pathogen-associated molecular patterns (PAMPs)2. This recognition triggers the formation of a phagocytic cup, where plasma membrane protrusions extend around the target particle3. Actin filaments play a crucial role in this step, driving the membrane remodeling necessary for particle engulfment4. Once the particle is enclosed in a phagosome, a membrane-bound vesicle is formed. The phagosome undergoes maturation, characterized by the fusion with early, then late endosomes, followed by the incorporation of lysosomes containing hydrolytic enzymes5. These enzymes, such as cathepsins, lysozyme, and nucleases, are activated by acidification within the phagosome, achieved by vacuolar ATPase (V-ATPase), lowering the pH to 4.5-5.5. The degradative process concludes with the exocytosis of remnants from the cell. Dysfunctions in this dynamic and complex process can result in significant immune challenges6,7,8.
To accurately evaluate the complex cellular process of phagocytosis, there is a need for improved and easy methods for monitoring the major cellular events of phagocytosis. Traditionally, monitoring method for phagocytosis quantification involve techniques such as fixing phagocytes at predetermined time intervals for microscopic visualization, measuring the internalization of dye-labeled targets through imaging or flow cytometry or counting the number of target cells remaining after a specified duration of phagocytosis, However, these methods do not capture the minute-to-minute fluctuations that occur during the phagocytosis process9. Moreover, methods relying on indirect indicators like dye uptake may not accurately reflect actual phagocytic events due to potential passive dye transfer10.
Recent advancements in live-cell imaging, flow cytometry, and staining techniques have revolutionized our ability to monitor phagocytosis with high spatiotemporal precision. Live-cell imaging, for instance, enables continuous observation of dynamic processes with sequential events tracking11. Flow cytometry is widely used for analyzing mammalian cells and detecting biomarkers in clinical research12. However, traditional FC methods are not equipped to evaluate the morphological and spatial characteristics of individual cells12. Imaging Flow Cytometry (IFC) serves as a robust alternative that enables the collection of detailed information from single cells. IFC combines the analytical capabilities of flow cytometry with high-resolution imaging, allowing single-cell analysis with morphological and spatial detail. Various particles, including fluorescent latex beads, zymosan-APC, and synthetic nanoparticles, are commonly used to study phagocytosis, providing insights into receptor engagement, phagosome formation, and immune responses12.
In contrast to conventional biochemical techniques, which typically rely on endpoint analysis, live-cell imaging provides continuous, dynamic monitoring of cellular processes. This method offers high spatiotemporal resolution and the ability to observe sequential events in phagocytosis. As a result, it is a powerful tool for studying the complex process of phagocytosis, allowing for more accurate and detailed insights into cellular behaviors and mechanisms13.
In addition to advancements in imaging systems, there is a need for smart phagocytic particles that can distinctly highlight phagocytic events, particularly the degradation steps occurring within the phagosome. Phagocytosis studies often utilize particles such as latex beads, zymosan, bacteria, apoptotic cells, and synthetic nanoparticles to explore mechanisms like receptor engagement, phagosome formation, and immune responses. While these particles provide valuable insights, they have limitations in tracking the complete degradation process or directly assessing phagosome maturation. For instance, latex beads are non-biodegradable and cannot demonstrate degradation, while zymosan and bacteria often require complex labeling and may not yield consistent degradation profiles14,15.
Our previous study demonstrated the utility of GB, a traditional Ayurvedic formulation composed primarily of calcium sulfate, as an ideal particle for studying phagosome maturation and degradation16. GB dissolves over time within the acidic environment of the phagosome, allowing clear visualization of particle degradation. Furthermore, GB&#39;s ability to induce robust vacuole formation facilitates the investigation of distinct stages of phagosome maturation, from early formation to acidification and eventual resolution. Unlike other particles, GB eliminates the need for external labeling, providing a self-sustained system to study the complete lifecycle of phagosomes, making it an ideal model for understanding the dynamics of phagocytosis and associated disorders16.
This study presents a standardized and reproducible protocol designed to evaluate particle-induced phagocytosis and vacuole formation using Godanti Bhasma (GB) in both professional (RAW 264.7) and non-professional (HeLa and 3T3-L1) phagocytic cell lines. The overall goal of this method is to provide a robust platform for studying cellular uptake mechanisms, vacuolar biogenesis, and intracellular particle processing across different cell types. GB was suspended in DMEM at a concentration of 10&#8239;mg/mL, and to ensure uniformity, larger particles were allowed to settle while the upper fraction was collected for experimental use. For flow cytometry assays, cells were seeded at a density of 2&#8239;x 105 cells per well in 12-well plates, and 300&#8239;&#181;L of the GB working suspension was added to each well. For imaging-based analyses, 5,000-10,000 cells were seeded per well in 96-well plates or on chamber slides. This method establishes GB as a reliable model for investigating vacuole formation and particle internalization in diverse cellular types.

<table><tbody><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; Culture Flasks</td><td>NEST, India</td><td>707001</td><td></td></tr><tr><td>96- and 6-well plates</td><td>Tarsons India Pvt. Ltd., Kolkata, India; Thermo Scientific, India</td><td>980040; 140675</td><td></td></tr><tr><td>Acridine orange</td><td>SRL, India</td><td>82407</td><td></td></tr><tr><td>Antibiotics, and Trypsin-EDTA solution</td><td>High Media, India</td><td>628242; 655111</td><td></td></tr><tr><td>Cell Lines- HeLa, 3T3-L1 and RAW-264.7</td><td>National Centre For Cell Science (NCCS), Pune, Maharastra</td><td></td><td></td></tr><tr><td>culture dishes (35mm)&amp;nbsp;</td><td>SPL Lifesciences, India</td><td>101350</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>MP Biomedicles, India</td><td>1033126</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>High Media, India</td><td>651257</td><td></td></tr><tr><td>Flow cytometer</td><td>Millipore, USA</td><td>Amnis</td><td>Amnis Imaging Flow Cytometers,</td></tr><tr><td>Fluorscent Microscope</td><td>DiGi Lab, India</td><td>Model: DiGi 110 Infinity</td><td></td></tr><tr><td>Godanti Bhasma</td><td>Patanjali Ayurved</td><td>240002</td><td></td></tr><tr><td>Neutral Red dye</td><td>CDH, India</td><td>593630</td><td></td></tr><tr><td>Time lapse microscope</td><td>NanoEnTek Inc.&amp;nbsp; Seoul, Korea.</td><td>JuLi</td><td>JuLi smart fluorescent cell analyzer,</td></tr><tr><td>Time lapse microscope</td><td>LEEDZ MICRO IMA. UK</td><td>S.No. LIM225552358A58</td><td>EA PRIME Inverted microscope</td></tr></tbody></table>

exploring the sequential cellular events of phagocytosis triggered by godanti bhasma in mammalian cells

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

Phagocytosis is a vital cellular mechanism through which cells engulf and degrade foreign particles, pathogens, or debris, playing a key role in immune defense and the maintenance of tissue homeostasis. Disruptions in this process are associated with various diseases. To explore the complex events involved in the phagocytosis pathway, advanced smart particles and effective monitoring techniques are essential. Godanti Bhasma (GB), a traditional Indian medicine composed of bioactive calcium sulfate particles, is rapidly internalized by phagocytosis in mammalian cells, inducing significant cytoplasmic vacuolation. The key stages of GB-induced phagocytosis were evaluated here using flow cytometry (FC), live-cell imaging, and specific staining techniques. Flow cytometric analysis demonstrated the formation of phagocytic cup-like structures associated with particle internalization. Live-cell imaging enabled real-time observation of phagocytic processes, including particle uptake, vacuole formation, degradation of engulfed materials, and vacuolar turnover. Staining with neutral red and acridine orange was employed to assess vacuolar acidification. Interestingly, treatment with the lysosomal inhibitor BFA1 in GB-treated cells did not lead to vacuolation, as evidenced by the lack of neutral red uptake, emphasizing the requirement for an acidic environment for vacuolation to occur. These findings underscore the potential of GB-induced phagocytosis as a model to elucidate the sequential cellular events involved in this process, which is critical for understanding host-pathogen interactions, intracellular trafficking, and developing innovative therapeutic strategies for disorders related to phagocytosis.

The study highlights key cellular responses to GB particles in mammalian cells. We used an imaging flow cytometer to investigate the internalization of GB particles in 3T3-L1 cells (Figure 1). The cells were exposed to particles for durations ranging from 30 min to 12 h. After 30 min, we observed the particles on the cell surface (Figure 1B), and the cell membrane began to fold inward at the attachment sites, indicating the initiation of internalization (<strong...

This study elucidates phagocytic processing of Godanti Bhasma (GB) particles in mammalian cells, characterizing their cellular uptake, vacuole dynamics, acidification, and degradation. These findings not only advance our understanding of fundamental phagocytosis mechanisms but also establish GB as a promising model system for developing novel therapeutic strategies.

Phagocytosis is a vital cellular mechanism through which cells engulf and degrade foreign particles, pathogens, or debris, playing a key role in immune ...

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.

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