Quantitative Analysis of Phagocytosis in Whole Blood Using Double Staining and Visualization

---

Elena V. Lysakova¹, Alexander N. Shumeev², Sergei A. Chuvpilo¹, Viktor S. Laktyushkin², Natalia A. Arsentieva³, Mikhail Yu. Bobrov¹, and Stanislav A. Rybtsov^2,a*

¹Immunobiology and Biomedicine Division, Center for Genetics and Life Sciences, Sirius University of Science and Technology, 354340 Sirius, Krasnodar Region, Russia

²Resource Center for Cell Technologies and Immunology, Sirius University of Science and Technology, 354340 Sirius, Krasnodar Region, Russia

³Saint-Petersburg Pasteur Institute, 197101 St. Petersburg, Russia

Received November 3, 2023; Revised January 9, 2024; Accepted February 19, 2024

---

Phagocytosis is an essential innate immunity function in humans and animals. A decrease in the ability to phagocytize is associated with many diseases and aging of the immune system. Assessment of phagocytosis dynamics requires quantification of bacteria inside and outside the phagocyte. Although flow cytometry is the most common method for assessing phagocytosis, it does not include visualization and direct quantification of location of bacteria. Here, we used double-labeled Escherichia coli cells to evaluate phagocytosis by flow cytometry (cell sorting) and confocal microscopy, as well as employed image cytometry to provide high-throughput quantitative and spatial recognition of the double-labeled E. coli associated with the phagocytes. Retention of pathogens on the surface of myeloid and lymphoid cells without their internalization was suggested to be an auxiliary function of innate immunity in the fight against infections. The developed method of bacterial labeling significantly increased the accuracy of spatial and quantitative measurement of phagocytosis in whole blood and can be recommended as a tool for phagocytosis assessment by image cytometry.

---

KEY WORDS: phagocytosis, E. coli, flow cytometry, confocal microscopy, human leukocytes

DOI: 10.1134/S0006297924050122

Publisher’s Note. Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

---