Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 5, pp. 923-932 © Pleiades Publishing, Ltd., 2024.

923

Quantitative Analysis of Phagocytosis in Whole Blood

Using Double Staining and Visualization

Elena V. Lysakova

, Alexander N. Shumeev

, Sergei A. Chuvpilo

Viktor S. Laktyushkin

, Natalia A. Arsentieva

, Mikhail Yu. Bobrov

and Stanislav A. Rybtsov

2,a

Immunobiology and Biomedicine Division, Center for Genetics and Life Sciences,

Sirius University of Science and Technology, 354340 Sirius, Krasnodar Region, Russia

Resource Center for Cell Technologies and Immunology,

Sirius University of Science and Technology, 354340 Sirius, Krasnodar Region, Russia

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

e-mail: rybtsov.sa@talantiuspeh.ru

Received November 3, 2023

Revised January 9, 2024

Accepted February 19, 2024

Abstract— Phagocytosis is an essential innate immunity function in humans and animals. A decrease in the abil-

ity 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 cytom-

etry (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.

DOI: 10.1134/S0006297924050122

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

Abbreviations: AF405,Alexa Fluor 405; DN,double-negative; Dim cells,cells whose fluorescence intensity is one order of

magnitude brighter than the intensity of DN cells; DP,double-positive for FITC and AF405; PBS,phosphate buffered saline.

* To whom correspondence should be addressed.

INTRODUCTION

Phagocytosis is common in animals and can be

found in protozoans and multicellular organisms.

Such mammalian phagocytic cells as (monocytes, mac-

rophages, neutrophils, dendritic cells) are components

of the innate immune system and the first line of de-

fense against a variety of pathogens that enter the

body and can cause dangerous infections. Phagocyto-

sis is also required for elimination of apoptotic and se-

nescent cells. Hence, phagocytosis is an indispensable

process for maintaining homeostasis in multicellular

animals [1]. A decrease in the ability of immune cells

to phagocytize is associated various disorders, includ-

ing organism’s immunodeficient state in infectious

diseases. Thus, reduced phagocytosis was found in the

periodontal disease [2], lower airway bacterial coloni-

zation [3], and other conditions leading to serious pa-

thologies [4,5].

Foreign agents invading the body are recognized

by “professional” phagocytes through a variety of Toll-

like receptors (TLRs) located on the surface of the

phagocyte. TLRs interact with bacterial cell wall compo-

nents, e.g., lipopolysaccharide (LPS– while Fc receptors

LYSAKOVA et al.924

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

recognize antibodies that opsonize pathogen’s anti-

gens [6]. Initiation of inflammation through the alter-

native mechanisms also primarily activates phagocyto-

sis. Phagocytosis is an essential and basic mechanism

that helps to eliminate a pathogen or to suppress its

spread. Therefore, many infections have evolutionari-

ly developed the pathways that provide phagocytosis

suppression as the main protective mechanism against

the host’s immunity [7, 8]. Inhibition of phagocytosis

is also one of the main mechanisms used by tumors to

escape immune recognition and removal [9].

Phagocytosis is an important link between innate

and adaptive immunity. Phagocytized pathogens are

processed into short peptides that are displayed on the

surface of phagocyte in the context of the histocompat-

ibility complex class  II (MHC  II) and are presented to

T  lymphocytes, thus initiating T  cell proliferation and

triggering adaptive immune response [10]. Therefore,

assessment of phagocytic activity is an extremely im-

portant diagnostic technique that can be used in pre-

venting immune pathologies, studying the mechanisms

of development of dangerous diseases, and testing

drugs that modulate innate immunity and phagocytosis.

The phagocytic activity of leukocytes is usually

assessed using special phagocytic tests [11,  12]. There

are multiple variations of this assay that use different

targets of phagocytosis. For example, Escherichia coli

is commonly employed as a safe and effective target

for phagocytosis in research tests. The cell wall of

E.  coli contains LPS, which is recognized by TLR4 lo-

cated on the surface of phagocyte. The phagocytic ac-

tivity can be assessed in both suspension and adhesive

(on plastic or extracellular matrix) fractions of blood

cells, as adhesion is not a requirement for phagocyto-

sis by neutrophils [13].

A number of studies have shown a decrease in the

phagocytic activity in people with age [14]. A decline

in the phagocytic activity is considered in conjunction

with other changes, e.g., decreased number of naïve

T and B cells, increased systemic production of anti-

inflammatory cytokines, reduced antigen presenta-

tion, and decreased secretion of specific antibodies.

Moreover, the age-related increase in the number

of myeloid cells is accompanied by a decrease in the

number of phagocytic cells in this population, as well

as a decrease in the number of lymphoid cells. These

phenomena are regarded as indications of the immune

system aging [15]. Age-related activation of myelopoi-

esis may be an adaptive mechanism compensating for

the loss of phagocytic function. Therefore, to accurate-

ly assess age-associated changes in the immune system

activity, it is necessary to develop quantitative meth-

ods for analyzing the dynamics of phagocytosis at the

level of individual cells.

An important quantitative parameter of phagocy-

tosis is the phagocytic number – the average number

of bacteria internalized by one phagocyte during the

experiment. However, being an integrated parameter,

the phagocytic number does not take into account the

spatial position of a bacterium relative to the phago-

cyte membrane, leading to the loss of information

on the kinetics of internalization and overestimation

of the indicators of phagocytosis. In addition, rou-

tine assessment of the phagocytic number requires

high-throughput methods for counting bacteria ad-

hered to the surface and phagocytized into the cells

in order to make a more accurate assessment of the

phagocytic activity that would take into account the

kinetics of the process.

Although microscopy can be used to estimate the

phagocytic number, it is time-consuming, which im-

poses limitations on the method performance and re-

producibility, as well as the statistical significance of

the results. Flow cytometry is a high-throughput pro-

cedure and one of the simplest and most accessible

methods for assessing the phagocytic activity, but its

use does not imply visualization of the obtained data,

which makes it difficult to quantitatively analyze the

spatial position of particles in a cell. Here, we present

a method developed to estimate the phagocytic num-

ber with simultaneous quantitative assessment of

E.  coli cells adhered to the surface and internalized by

phagocytes using flow cytometry.

The method developed involves sequential con-

jugation of fixed bacterial cells with fluorescein-5-iso-

thiocyanate (FITC) and biotin. After incubation with

human peripheral blood, remaining external (adher-

ent) bacteria are detected by staining with Alexa Fluor

(AF405) conjugated with streptavidin. Streptavidin

does not penetrate into phagocytes and selectively in-

teracts with biotin covalently bound to proteins on the

surface of E.  coli cells. Therefore, the ingested bacte-

ria would be labeled with FITC only, while externally

attached bacteria would be positive for both AF405 and

FITC (double-positive, DP), which allows to determine

the location of each E.  coli bacterium. When analyzed

by flow cytometry, each individual cell was assessed

for the fluorescence signal intensity, which allowed to

distinguish several cell subpopulations and to suggest

the existence of phagocytes with different numbers

of internalized bacteria already at the initial stage of

study.

We combined the data from flow cytometry and

cell sorting with confocal microscopy imaging for quan-

tification of phagocytized bacteria, thus validating the

use of flow cytometry in routine studies requiring no

visualization.

In addition to the time-consuming analysis by a

combination of cell sorting and visual quantification

by confocal microscopy, the samples were tested using

an Amnis Flowsight imaging flow cytometer to accel-

erate the assessment of the spatial location of bacteria

QUANTITATIVE ANALYSIS OF PHAGOCYTOSIS 925

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

in the cells. The double-staining procedure developed

in this study significantly improved the accuracy of

analysis of phagocytosis of bacterial cells using mod-

ern imaging flow cytometers.

MATERIALS AND METHODS

Conjugation of bacteria with FITC and biotin.

Sterile glycerol (final concentration, 10%) was added to

20  ml of E.  coli overnight culture (strain DH5a) grown

from a single colony in LB medium and the resulting

suspension was frozen at –20°C in 50-µl aliquots. One

day before the experiment, 50  μl of the E.  coli stock

suspension in 10% glycerol was transferred to 10  ml

of LB medium and grown overnight. The optical den-

sity of the cell suspension was determined with a Mul-

tiskan SkyHigh Microplate Spectrophotometer (Thermo

Fisher, USA) at 540  nm. E.  coli cells were diluted with

LB medium to an optical density of 0.6, which cor-

responded to a concentration of 1.29  ×  10

bacteria

per ml (according to a previously developed calibra-

tion curve). Next, 500  μl of E.  coli cell suspension was

centrifuged for 1  min at 2350g. The pellet was resus-

pended in 250  μl of phosphate buffered saline (PBS),

250  μl of fixative (10% neutral buffer formalin, El-

ement Company, Russia) was added, and the bacte-

rial suspension was thoroughly mixed by pipetting

and vortexing. E.  coli cells were fixed for 30  min at

room temperature on a rotary mixer (10  rpm), washed

3 times with 500  μl of PBS, and pelleted by centrifu-

gation for 2 min at 2350g. After the last centrifuga-

tion, the pellet was resuspended in 250μl of PBS with

250  μl of borate buffer (50  mM, pH  9.0) and labeled

with FITC (Lumiprobe, Russia) according to the manu-

facturer’s instructions. For this purpose, dry FITC was

dissolved at a concentration of 20  mg/ml in dimethyl

sulfoxide, aliquoted into 10  μl, and stored at –80°C un-

til use. Prepared FITC stock solution (1  μl) was added

to 500  μl of bacterial suspension, and immediately

mixed vigorously. The cell suspension was incubated

for 16  h in the dark at 37°C with constant shaking in

a Biosan TS-100 thermoshaker (400  rpm). After incu-

bation, bacterial cells were washed three times with

500  μl of PBS and pelleted by centrifugation for 2  min

at 2350g. After the last centrifugation, the pellet was

resuspended in 200  μl of PBS; the cells were counted

and stored at 4°C for a maximum of 24  h before use.

Alternatively, the cells were resuspended in 200  μl of

10%  glycerol solution, stored in aliquots at –20°C, and

used for conjugation with FITC only. The efficiency of

conjugation was assessed with a BD LSRFortessa flow

cytometer from the fluorescence signal intensity in the

FITC channel (488-nm laser; filter, 530/20  nm) in com-

parison with the control unconjugated fixed bacteria

(Fig.1). Next, FITC-labeled bacterial cells were conju-

gated with biotin using a FluoReporter Mini-biotin-XX

Protein Labeling Kit (Thermo Fisher; cat. no. F6347)

according to the manufacturer’s instructions. Briefly,

200  μl of bacterial suspension was mixed with 20  μl of

freshly prepared 1  M solution of sodium bicarbonate

in water (pH  8.3-8.5) and 20

  μl of active biotin ester

(Component  A) solution in deionized water was added

(200  μl of water was added to a tube with dry Com-

ponent  A immediately before use, since the reactive

form of biotin is quickly hydrolyzed in water). Bacteri-

al cells were incubated with biotin for 2  h in the dark

at room temperature at constant stirring in a Biosan

TS-100 thermoshaker (400  rpm), washed twice with

PBS by centrifugation for 2  min at 2350g, and resus-

pended in 200  μl of PBS. Sterile glycerol was added to

a final concentration of 10%. After the concentration

of labeled bacteria was determined using a cell-count-

ing chamber and adjusted with PBS to 10

  cell/ml, the

cells aliquoted in 20  μl, frozen, and stored at –20°C. All

procedures were carried out under sterile conditions.

To test the efficiency of biotinylation procedure,

1  μl of streptavidin conjugated with AF405 (Invitrogen,

USA, cat. no.S32351) was added to an aliquot of E.  coli

cells (300  μl, 1×10

E. coli/ml) and the mixture was in-

cubated on ice for 30  min. The cells were washed with

1.2  ml of PBS, centrifuged for 2  min at 2350g, resus-

pended in 200  μl of PBS, and analyzed on a BD LSR-

Fortessa flow cytometer to assess the DP cell popu-

lation in the FITC and AF405 channels (405-nm laser,

450/50  nm filter) (Fig.1).

All solutions were prepared in deionized water

(18.2  MΩ·cm) produced by a Millipore Milli-Q IQ7000

water purification system (Merck, USA).

Phagocytic test. Venous blood was collected from

healthy volunteers using a system for vacuum blood

collection into the tubes containing heparin sodium

(Khimmedsnab, Russia) at the Sirius University Med-

ical Center by a qualified personnel.

The concentration of leukocytes in the sample was

determined using a Celltac MEK-7300K hematology an-

alyzer (Nihon Kohden, Japan). This information was

further used to calculate the amount of added bac-

teria based on the leukocyte  :  bacteria ratio of 1  :  20,

in accordance with previously published works [16].

For the test, 100  μl of whole blood was pipetted into

a polypropylene non-adhesive tube (Eppendorf, Ger-

many, cat. no. 0030125150) and its temperature was

stabilized in a CO

incubator (37°C, 5%  CO

) for 5  min.

The phagocytic test was carried out in the suspension

fraction of whole blood cells without using special pro-

cedures for cell adhesion to the surface [13]. Bacterial

suspension was added to 100  μl of blood at a ratio of

20 bacterial cells per leukocyte and incubated for 1  h

at 37°C in the CO

incubator. To stop phagocytosis, the

samples were placed on ice (Fig.  S1 in the Online Re-

source1), [17].

LYSAKOVA et al.926

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

Fig. 1. Scheme of E. coli cell labeling (conjugation and quality control). Fixed bacterial cells were sequentially conjugated with

FITC and biotin to be used for determining their spatial location inside and outside of phagocytes. The concentration of bacteria

was determined by measuring the optical density(a) using a pre-calculated calibration curve(b). After conjugation with FITC,

the uniformity of conjugation was verified by flow cytometry; 99.7% cells contained the FITC label(c). After subsequent con-

jugation with biotin, the uniformity of labeling was verified in both streptavidin-AF405 and FITC channels; the uniformity of

conjugation was 98.7% for both labels(d). Before freezing the stock, the concentration of labeled bacteria was determined by the

differential interference contrast (DIC) and fluorescence methods using a Countess3 automated cell counter (Thermo Fisher).

Flow cytometry and cell sorting. Anti-CD45 an-

tibody APC-eFluor780 (clone HI30, eBioscience, USA,

cat. no. 47-0459-42; 1  :  100) was added to an aliquot

of blood on ice after phagocytosis to label leuko-

cytes; AF405-conjugated streptavidin (Invitrogen, cat.

no.S32351; 1  :  100) was added to detect bacteria on the

cell surface. The samples were incubated for 30  min

in the dark on ice, washed with 1.4  ml of PBS, and

centrifuged for 5  min at 330g. The supernatant was

discarded and the pellet was resuspended by gentle

pipetting in 100  μl of PBS; next, 900  μl of 1×  BD FACS

Lysing Solution (BD, cat. no. 349202) was added and

the samples were incubated for 10  min in the dark at

room temperature and then centrifuged for 5  min at

330g. The pellet was resuspended in 200  μl of PBS and

stored at 4°C until analysis (see Fig. S1 in the Online

Resource1).

The compensation setup was carried out in an

automated mode for a flow cytometer or a cell sorter

using single-stained controls prepared as described

above except the control for AF405, which was pre-

pared by staining an aliquot of blood with biotin-

conjugated anti-CD33 antibodies (clone AC104.3E3,

Miltenyi; cat. no. 130-113-347) for 30 min on ice, fol-

lowed by treatment with AF405-conjugated streptavi-

din and further processing as described above.

Preliminary analysis of the samples was carried

out on a BD LSRFortessa flow cytometer. To deter-

mine the number of E.  coli cells and their spatial lo-

cation in white blood cells after phagocytosis, the cells

were sorted with a Sony SH800 sorter equipped with

4 lasers (405, 488, 561, and 638 nm) and 100-micron

nozzle chips and analyzed with a confocal microscope.

The gaiting strategy implied sequential identification

of single cells for the forward scatter (FSC) and then

for the side scatter (SSC) according to the Area and

Height pulse parameters, removal of cell debris based

on the lighting scattering parameters, isolation of

CD45

population, and identification of subpopulations

of phagocytic leukocytes based on the fluorescence

QUANTITATIVE ANALYSIS OF PHAGOCYTOSIS 927

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

signal intensity in the FITC and AF405 channels (Fig. S2

in the Online Resource  1). The cells were sorted into

5-ml polystyrene tubes with 1  ml of PBS (at least 10,000

cells for each leukocyte subpopulation).

Next, we used high-performance flow cytometry

with visualization to determine the spatial location

and number of bacteria inside and outside leukocytes.

The work was performed using an Amnis FlowSight

imaging flow cytometer (Cytek, USA) equipped with

three lasers (405, 488, and 642nm) that was provided

by the Cytometry and Biomarkers Core Facility at the

St. Petersburg Pasteur Research Institute of Epidemi-

ology and Microbiology.

Confocal microscopy. After sorting, the cells

were concentrated by centrifugation for 5  min at 330g,

resuspended in 10μl PBS and placed on charged glass

slides (Polysine Adhesion Slides, Thermo Fisher). The

slides were incubated in a humid chamber at room

temperature for 30 min, covered with coverslips, and

analyzed under an inverted laser scanning confocal

ZEISS LSM 980 Airyscan microscope equipped with

a 20×  lens (Plan-Apochromat 20×, numerical aper-

ture 0.8). The images in 4 channels were obtained by

sequential scanning of two tracks: APC-eFluor 780

channel in the first track and AF405, FITC, and DIC

(differential interference contrast) channels in the sec-

ond track. APC-eFluor 780 was excited with a 639-nm

laser [maximum power, 20  mV; acousto-optic tunable

filter (AOTF) throughput, 2.6%] with signal detection

at 647 to 757  nm (detector type, GaAsP; amplification,

713  V). AF405 was excited with a 405-nm laser (maxi-

mum power, 20  mV; AOTF throughput, 0.2%) with sig-

nal detection at 410 to 484  nm (detector type, GaAsP;

amplification, 650  V). FITC was excited with a 488-nm

laser (maximum power, 13  mV; AOTF throughput,

0.04%) with signal detection at 519 to 628  nm (detector

type, Multialkali-PMT; amplification, 845  V). DIC imag-

es in transmitted light were obtained using the second

track lasers (405 and 488 nm) with a T-PMT detection

at 300 to 900  nm (Multialkali-PMT detector; amplifica-

tion, 368  V). The following scanning parameters were

used: scanning zoom, 8×; image size, 429×429 pixel;

pixel time (signal accumulation time), 4.91 microsec-

ond; pixel size, dx  =  dy  = 0.124 μm. The images were

obtained using the ZEN Blue3.2 (Zeiss) software.

Software. The results were processed in Micro-

soft Excel 2019. Flow cytometry data were analyzed

with BD FlowJo v. 10.9.0; Amnis FlowSight data were

analyzed with Amnis IDEAS 6.2; confocal microscopy

data were analyzed with ImageJ/Fiji v.1.54f [18].

RESULTS AND DISCUSSION

Quantitative analysis of phagocytosis by cell

sorting and microscopy. After phagocytosis, several

subpopulations of CD45

leukocytes were gated by the

granularity, size and E.  coli fluorescence (Fig. 2). Toan-

alyze the distribution of internalized or surface-ad-

hered bacteria in granulocytes, monocytes, and lym-

phocytes, these cell populations were sorted with a

Sony SH800 sorter (see Fig.2 and Fig.S2 in the Online

Resource1 for the gating strategy) and analyzed under

a ZEISS LSM 980 Airyscan confocal microscope. After

establishing the protocol for the procedure, cell sort-

ing was carried out three times using blood samples of

three donors.

Comparison of cell sorting and microscopy data is

presented in Fig.  3. The most abundant population was

a double-negative (DN) subpopulation (71.2% of all

CD45

cells). None of these cells contained E.  coli bacte-

ria inside or on the surface, as was found by the analy-

sis of 74 confocal microscopy images of the sorted DN

cells from three donors. DIM cells (cells whose fluores-

cence intensity was 10 times brighter than the fluores-

cence of DN cells) demonstrated dull fluorescence in

the FITC channel. Out of 33 DIM cells analyzed under

a microscope, only one contained bacteria; all other

cells had a slightly fluorescent cytoplasm that was like-

ly a background autofluorescence resulting from the

uptake of the debris of FITC-conjugated bacteria. The

MID subpopulation forms a distinct cluster with FITC

fluorescence approximately 10 times brighter than the

fluorescence of DIM cells. The fluorescence intensity

of MID cells in the AF405 channel corresponded to the

autofluorescence of DN cells. This indicates that the

MID population consisted of cells that have phagocy-

tized bacteria and did not contain E.  coli on the surface.

Analysis of confocal microscopy images (total of

151 cells) confirmed this assumption. The phagocytic

number in the MID population varied from 1 to 5 (av-

erage value, 1.87 bacteria per cell). DP cells (3% of all

cells) contained bacteria either on the surface only or

on the surface and inside the cells (Fig.  3). The average

number of bacteria inside the cells in the DP population

(based on analysis of 51 cells) was 0.67 bacteria per cell

(value range, 0-16); the average number of bacteria

outside the cells was 1.41 (value range, 1-6). Hence, the

DP population was extremely heterogeneous and can

be further divided into several subpopulations.

Visualization of phagocytes in the flow using

double staining revealed the presence of a signif-

icant amount of E. coli on the surface of the cells.

Although sequential use of cell sorting and confocal

microscopy allows to assess the quantity and spatial

location of bacteria in phagocytes, such analysis is la-

borious and time-consuming. The use of flow cytome-

try only makes it possible to estimate the number of

E.  coli bacteria inside the phagocytes based on fluo-

rescence, but does not allow to quantify phagocytosis

when the bacteria are located simultaneously inside

the cells and on their surface.