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Peer-reviewedCC BY 4.0Molecules

Activated CaSO4-Induced Vacuolation as a Quantitative Platform for Phagocytosis-Driven Drug Screening

  • Vishakha Goswami1
  • Abul Faiz1
  • Gaurav Dutt1
  • Saima Bashir1
  • Arushi Gupta1
  • Snehasish Das1
  • Apurba Gouri2
  • Alpana Joshi1
  • Subrata K. Das1
  1. 1School of Biomedical Engineering and Health Sciences, Shobhit Institute of Engineering and Technology, NH 58, Meerut 250110, India
  2. 2Department of Chemistry, Jeonbuk National University, Jeonju 54896, Republic of Korea
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Abstract

Cytoplasmic vacuolization is associated with phagocytosis, lysosomal acidification, and autophagy, yet robust in vitro models for its quantification and pharmacological screening remain limited. Here, we demonstrate that thermally activated calcium sulfate (ACS) induces extensive vacuolization across mammalian cell lines, including HeLa, RAW 264.7, 3T3-L1, and SH-SY5Y, establishing a versatile platform for studying vacuole biogenesis. To improve reproducibility, ACS particle heterogeneity was addressed by sedimentation-based fractionation, with the 5 min fraction producing homogeneous suspensions and stable, consistent vacuole formation. Vacuolization was quantified using Neutral Red (NR) uptake, and dose- and time-dependent analyses demonstrated a direct relationship between ACS concentration and vacuole induction. The assay was validated using bafilomycin A1 (BFA1), a selective V-ATPase inhibitor, which demonstrated concentration- and time-dependent inhibition of vacuole formation and acidification. Screening of ten commercially available drugs revealed distinct responses, including early cytotoxicity, strong vacuole inhibition, partial suppression, or negligible effects. The ability to distinguish vacuole inhibition from cytotoxic responses highlights the utility of ACS-induced vacuolization as a sensitive and scalable in vitro platform for mechanistic studies and functional screening of modulators of lysosomal and phagocytic pathways relevant to infection, lysosomal disorders, and phagocytic dysfunction.

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Cite

BibTeXRIS
BibTeX
@article{goswami2026activated,
  title = {{Activated CaSO4-Induced Vacuolation as a Quantitative Platform for Phagocytosis-Driven Drug Screening}},
  author = {Vishakha Goswami and Abul Faiz and Gaurav Dutt and Saima Bashir and Arushi Gupta and Snehasish Das and Apurba Gouri and Alpana Joshi and Subrata K. Das},
  journal = {Molecules},
  year = {2026},
  month = nov,
  doi = {10.3390/molecules31214022},
  url = {https://www.mdpi.com/1420-3049/31/21/4022},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Activated CaSO4-Induced Vacuolation as a Quantitative Platform for Phagocytosis-Driven Drug Screening
AU  - Vishakha Goswami
AU  - Abul Faiz
AU  - Gaurav Dutt
AU  - Saima Bashir
AU  - Arushi Gupta
AU  - Snehasish Das
AU  - Apurba Gouri
AU  - Alpana Joshi
AU  - Subrata K. Das
PY  - 2026
DA  - 2026/11/08
JO  - Molecules
DO  - 10.3390/molecules31214022
UR  - https://www.mdpi.com/1420-3049/31/21/4022
AB  - Cytoplasmic vacuolization is associated with phagocytosis, lysosomal acidification, and autophagy, yet robust in vitro models for its quantification and pharmacological screening remain limited. Here, we demonstrate that thermally activated calcium sulfate (ACS) induces extensive vacuolization across mammalian cell lines, including HeLa, RAW 264.7, 3T3-L1, and SH-SY5Y, establishing a versatile platform for studying vacuole biogenesis. To improve reproducibility, ACS particle heterogeneity was addressed by sedimentation-based fractionation, with the 5 min fraction producing homogeneous suspensions and stable, consistent vacuole formation. Vacuolization was quantified using Neutral Red (NR) uptake, and dose- and time-dependent analyses demonstrated a direct relationship between ACS concentration and vacuole induction. The assay was validated using bafilomycin A1 (BFA1), a selective V-ATPase inhibitor, which demonstrated concentration- and time-dependent inhibition of vacuole formation and acidification. Screening of ten commercially available drugs revealed distinct responses, including early cytotoxicity, strong vacuole inhibition, partial suppression, or negligible effects. The ability to distinguish vacuole inhibition from cytotoxic responses highlights the utility of ACS-induced vacuolization as a sensitive and scalable in vitro platform for mechanistic studies and functional screening of modulators of lysosomal and phagocytic pathways relevant to infection, lysosomal disorders, and phagocytic dysfunction.
SN  - 1420-3049
ER  -