Our laboratory has developed a flow cytometric assay to quantify alveolar macrophage (MФ) phagocytosis of bacteria within a live animal. MФs collected by bronchoalveolar lavage from rats infected transtracheally with Syto 9-labeled bacteria are fluorescently labeled for identification and analyzed by flow cytometry to quantify their bacterial uptake

Gentry-Nielsen, Martha J.

Jensen-Smith, Heather C.

Perry, Greg A.

Pitz, Adam M.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Department of Veterans Affairs Staff Publications U.S. Department of Veterans Affairs 2010 A flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophages Adam M. Pitz Creighton University School of Medicine Greg A. Perry Creighton University School of Medicine, gperry@creighton.edu Heather C. Jensen-Smith Creighton University School of Medicine Martha J. Gentry-Nielsen Creighton University School of Medicine, mgentry@creighton.edu Follow this and additional works at: https://digitalcommons.unl.edu/veterans Pitz, Adam M.; Perry, Greg A.; Jensen-Smith, Heather C.; and Gentry-Nielsen, Martha J., "A flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophages" (2010). U.S. Department of Veterans Affairs Staff Publications. 65. https://digitalcommons.unl.edu/veterans/65 This Article is brought to you for free and open access by the U.S. Department of Veterans Affairs at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in U.S. Department of Veterans Affairs Staff Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. NoteA flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophagesAdam M. Pitz a, Greg A. Perry a, Heather C. Jensen-Smith b, Martha J. Gentry-Nielsen a,c,⁎a Department of Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USAb Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USAc Research Service, Omaha Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105, USAa b s t r a c ta r t i c l e i n f oArticle history:Received 11 September 2009Received in revised form 8 February 2010Accepted 11 February 2010Available online 18 February 2010Keywords:Animal ModelsHost DefenseHumanLungPhagocytosisOur laboratory has developed a flow cytometric assay to quantify alveolar macrophage (MФ) phagocytosis ofbacteria within a live animal. MФs collected by bronchoalveolar lavage from rats infected transtracheallywith Syto 9-labeled bacteria are fluorescently labeled for identification and analyzed by flow cytometry toquantify their bacterial uptake.© 2010 Elsevier B.V. All rights reserved.Recent studies have utilized flow cytometry to measure macro-phage (MΦ) phagocytosis of microorganisms (Stojkovic et al., 2008;Luo et al., 2006; Gille et al., 2006; Shida et al. 2006; Utaisincharoenet al., 2003). However, quantifying alveolar MФ phagocytosis byflow cytometry is problematic due to their high autofluorescence.Additionally, MΦ phagocytosis assays performed in vitro with pre-opsonized or modified (e.g. heat-killed) bacteria to facilitate uptake(Medeiros et al., 2009; Fitzpatrick et al., 2008; Brown et al., 2007;Kudo et al., 2004) are limited by not adequately mimicking thebiological environment within the infected lung. We thereforedeveloped an in vivo assay using live, non-opsonized organisms thatallows examination of uptake within the lungs as well as detection ofunderlying host immune defects leading to reduced opsonization and/or phagocytosis during pulmonary infections. Our assay describedherein has the potential for adaptation to other pathogens, otheranimal models, and even in vitro human studies.Staphylococcus aureus 29213, Streptococcus pneumoniae 6303,Klebsiella pneumoniae 35657, and Escherichia coli 25922 (AmericanType Culture Collection, Rockville, MD, USA) were grown on sheepblood agar plates (Remel, Lenexa, KS, USA) for 16 hours at 37 °C in 5%CO2. Organisms suspended in sterile water to an optical density of 1.0 at540 nm (∼3×108 colony forming units (cfu)/ml) were incubated atroom temperature for 15 min in the dark with 10 nM Syto 9 (MolecularProbes, Eugene, OR, USA). They were washed twice to remove excessdye and resuspended in sterile water. A FACSAria cytometer (BectonDickinson, San Jose, CA, USA)with a 530/30 band pass filter was utilizedto detect Syto 9 fluorescence. Syto 9 labeled staphylococci effectively,causing them to fluoresce more intensely than the autofluorescence ofrat alveolar MΦs (Fig. 1A). Syto 9 did not alter the viability of thebacteria, as shown by comparison of plate counts performed before andafter staining (7×108 cfu/ml vs. 5×108 cfu/ml, respectively). Todemonstrate that our assay could be adapted to measure phagocytosisof other common bacteria, we used Syto 9 to directly stain S.pneumoniae, K. pneumoniae, and E. coli, all of which stained morebrightly than rat MΦs (Fig. 1B-D). Several other fluorescent dyes wereevaluated, but failed due to leakage (DAPI and Hoechst 33258), lowfluorescent intensity (CT Blue CMAC and Qdot 800) or the need forspecies-specific reagents (APC-Cy7).Pathogen-free, male Sprague-Dawley rats (Charles River Labora-tories, Kingston, NY, USA) were infected transtracheally with1×108 cfu of Syto 9-labeled staphylococci under light anesthesia asdescribed previously (Vander Top et al., 2006). To prevent the loss ofbacterial fluorescent intensity due to bacterial lysis within the MФs,the rats were euthanized exactly 15 minutes post-infection by anintraperitoneal injection of 75 mg/kg body weight of pentobarbital(Nembutal, Abbott Laboratories, Abbott Park, IL, USA) followed byexsanguination. Ex vivo bronchoalveolar lavage was performed tocollect pulmonary cells from the rats’ lungs as described previously(Preheim et al., 1991). The recovered cells were stained withphycoerythrin (PE)-conjugated antibody (RP-1; BD Pharmingen,San Diego, CA, USA) to label neutrophils and a biotinylated antibodyJournal of Microbiological Methods 81 (2010) 194–196⁎ Corresponding author. Department of Microbiology and Immunology, CreightonUniversity School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.Tel.: +1 402 280 2346; fax: +1 402 280 1875.E-mail address: mgentry@creighton.edu (M.J. Gentry-Nielsen).0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.mimet.2010.02.005Contents lists available at ScienceDirectJournal of Microbiological Methodsj ourna l homepage: www.e lsev ie r.com/ locate / jmicmeth(1C7; BD Pharmingen) followed by streptavidin-allophycocyanin(APC) (BD Pharmingen) to label MФs (Vander Top et al., 2006).Three-color flow cytometric analysis was performed using duallaser excitation (488 nm and 637 nm). On each assay day, MФscollected from a control rat infected with non-fluorescent staphylo-cocci were used to exclude autofluorescence in the Syto 9 channel.Based on scatter characteristics, a minimum of 10,000 cells wereanalyzed in the APC (660 nm) channel for MФs and PE (575 nm)channel for neutrophils. The percentage of MФs with associatedbacteria was determined as the percentage of APC positive cellsfluorescing brighter in the Syto 9 (530 nm) channel than MФs fromthe control rat, as analyzed using FlowJo Software (Tree Star, Ashland,OR, USA).Confocal microscopy was performed to ensure that staphylococciin positive MΦs had been internalized rather than bound to the cellsurface. Cytospin slides prepared from lavage samples of each ratwere examined with a scanning confocal microscope (Zeiss Micro-Imaging, Thornwood, NY, USA). Syto 9-labeled staphylococci wereimaged using 488 nm excitation and a band pass filter of 500-530 nm.A Chameleon ULTRA titanium:sapphire laser (Coherent, Santa Clara,CA, USA) was used for two-photon excitation (760 nm) of DAPI-stained MФ nuclei (435-485 nm band pass filter). Syto 9-labeledstaphylococci were observed throughout MФs that were opticallysectioned at 0.82 μm intervals. Individual MФ images in the samefocal plane as MФ nuclei indicated the presence or absence ofcytoplasmic bacteria as depicted in Figs. 2A and B, respectively. Fewerthan 2% of the positive cells demonstrated 1-2 surface-associatedbacteria, verifying that the uptake measured was from phagocytosedorganisms.Manual counts of cytospin slides stained with hematoxylin andeosin also were performed to confirm the MΦ phagocytosis valuesdetermined by flow cytometry. Two hundred cells from each samplewere counted independently by two technicians using a lightmicroscope to quantify the percentage of MФs containing phagocy-tosed staphylococci (Fig. 2C) vs. those without internalized bacteria(Fig. 2D). The flow cytometry and light microscopy phagocytosisvalues from 17 rats were compared by regression analysis (Fig. 3). Themean percent of MΦ phagocytosis determined by flow cytometry was23.5% and by manual counting was 16.6%, with a Spearman's rankorder correlation coefficient of 0.63 (p=0.007). Manual counts ofMФs from control rats infected with unstained bacteria also weresimilar, indicating that Syto 9 did not affect staphylococcal virulenceor adherence.In conclusion, we have developed a flow cytometric assay toquantify bacterial uptake by MФs in vivo. The in vivo assay allows thedetermination to be made within the lungs of experimental animalsduring an actual infection. Although the assay was developed with S.aureus in rat lungs, successful staining of additional bacterial speciesindicates it can be adapted to other microorganisms and alternative invivo animal models, as well as to quantification of uptake by humanalveolar MФs in vitro.The authors would like to thank Mary Snitily for her skilledtechnical assistance in development of the assay. This research wassupported by revenue from Nebraska cigarette taxes awarded toFig. 1. Syto9-labeledbacteria. S aureus (A), S. pneumoniae (B),K. pneumoniae (C), andE. coli (D)were successfully labeledwith Syto 9andflourescedmore intensely thanMФ autofluorescence.195A.M. Pitz et al. / Journal of Microbiological Methods 81 (2010) 194–196Creighton University by the Nebraska Department of Health andHuman Services. It was performed with resources and the use offacilities at the Omaha Veterans Affairs Medical Center and wasapproved by the VA Animal Use and Care Committee. Confocalmicroscopy was conducted at the Integrative Biological ImagingFacility at Creighton University, which is supported by CreightonUniversity School of Medicine and constructed with support from C06Grant RR17417-01 from the NCRR, NIH.ReferencesBrown, L.A., Ping, X.D., Harris, F.L., Gauthier, T.W., 2007. Glutathione availabilitymodulates alveolar macrophage function in the chronic ethanol-fed rat. Am. J.Physiol. Lung. Cell. Mol. Physiol. 292, L824–L832.Fitzpatrick, A.M., Holguin, F., Teague, W.G., Brown, L.A., 2008. Alveolar macrophagephagocytosis is impaired in children with poorly controlled asthma. J. Allergy. Clin.Immunol. 121, 1372–1378.Gille, C., Spring, B., Tewes, L., Poets, C.F., Orlikowsky, T., 2006. A newmethod to quantifyphagocytosis and intracellular degradation using green fluorescent protein-labeledEscherichia coli: comparison of cord blood macrophages and peripheral bloodmacrophages of healthy adults. Cytometry 69, 152–154.Kudo, K., Sano, H., Takahashi, H., Kuronuma, K., Yokota, S., Fujii, N., Shimada, K., Yano, I.,Kumazawa, Y., Voelker, D.R., Abe, S., Kuroki, Y., 2004. Pulmonary collectins enhancephagocytosis of Mycobacterium avium through increased activity of mannosereceptor. J. Immunol. 172, 7592–7602.Luo, Y., Cook, E., Fries, B.C., Casadevall, A., 2006. Phagocytic efficacy of macrophage-likecells as a function of cell cycle and Fcgamma receptors (FcgammaR) andcomplement receptor (CR)3 expression. Clin. Exp. Immunol. 145, 380–387.Medeiros, A.I., Serezani, C.H., Lee, S.P., Peters-Golden, M., 2009. Efferocytosis impairspulmonary macrophage and lung antibacterial function via PGE2/EP2 signaling.J. Exp. Med. 206, 61–68.Preheim, L.C., Gentry, M.J., Snitily, M.U., 1991. Pulmonary recruitment, adherence, andchemotaxis of neutrophils in a rat model of cirrhosis and pneumococcalpneumonia. J. Infect. Dis. 164, 1203–1206.Shida, K., Kiyoshima-Shibata, J., Nagaoka, M., Watanabe, K., Nanno, M., 2006. Inductionof interleukin-12 by Lactobacillus strains having a rigid cell wall resistant tointracellular digestion. J. Dairy. Sci. 89, 3306–3317.Stojkovic, B., Torres, E.M., Prouty, A.M., Patel, H.K., Zhuang, L., Koehler, T.M., Ballard, J.D.,Blanke, S.R., 2008. High-throughput, single-cell analysis of macrophage interac-tions with fluorescently labeled Bacillus anthracis spores. Appl. Environ. Microbiol.74, 5201–5210.Utaisincharoen, P., Kespichayawattana, W., Anuntagool, N., Chaisuriya, P., Pichyangkul,S., Krieg, A.M., Sirisinha, S., 2003. CpG ODN enhances uptake of bacteria by mousemacrophages. Clin. Exp. Immunol. 132, 70–75.Vander Top, E.A., Perry, G.A., Gentry-Nielsen, M.J., 2006. A novel flow cytometric assay formeasurement of in vivo pulmonary neutrophil phagocytosis. BMC. Microbiol. 6, 61.Fig. 2.Microscopic images ofMФswith andwithout bacteria. A 0.82 µm thick optical slice of aMФ displaysfluorescent puncta of varying sizes indicating the presence of internalized Syto-9-labeled bacteria in multiple cytoplasmic foci (A) compared to a MФ without bacteria (B). As a comparison to flow cytometry, two hundred cells from each rat were counted by lightmicroscopy to determine the percentage of MФs that phagocytosed bacteria. A MФ that ingested bacteria (C) was easily identified from aMФ that had no bacteria (D). Scale bar=5 µm.Fig. 3. Comparison of MФ phagocytosis values as determined by flow cytometry andlight microscopy. A strong correlation exists between both values (p=0.007).196 A.M. Pitz et al. / Journal of Microbiological Methods 81 (2010) 194–196

A flow cytometric assay to quantify \u3ci\u3ein vivo\u3c/i\u3e bacterial uptake by alveolar macrophages

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University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
U.S. Department of Veterans Affairs Staff 
Publications U.S. Department of Veterans Affairs 
2010 
A flow cytometric assay to quantify in vivo bacterial uptake by 
alveolar macrophages 
Adam M. Pitz 
Creighton University School of Medicine 
Greg A. Perry 
Creighton University School of Medicine, gperry@creighton.edu 
Heather C. Jensen-Smith 
Creighton University School of Medicine 
Martha J. Gentry-Nielsen 
Creighton University School of Medicine, mgentry@creighton.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/veterans 
Pitz, Adam M.; Perry, Greg A.; Jensen-Smith, Heather C.; and Gentry-Nielsen, Martha J., "A flow cytometric 
assay to quantify in vivo bacterial uptake by alveolar macrophages" (2010). U.S. Department of Veterans 
Affairs Staff Publications. 65. 
https://digitalcommons.unl.edu/veterans/65 
This Article is brought to you for free and open access by the U.S. Department of Veterans Affairs at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in U.S. Department of Veterans 
Affairs Staff Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Note
A flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophages
Adam M. Pitz a, Greg A. Perry a, Heather C. Jensen-Smith b, Martha J. Gentry-Nielsen a,c,⁎
a Department of Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA
b Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA
c Research Service, Omaha Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105, USA
a b s t r a c ta r t i c l e i n f o
Article history:
Received 11 September 2009
Received in revised form 8 February 2010
Accepted 11 February 2010
Available online 18 February 2010
Keywords:
Animal Models
Host Defense
Human
Lung
Phagocytosis
Our laboratory has developed a flow cytometric assay to quantify alveolar macrophage (MФ) phagocytosis of
bacteria within a live animal. MФs collected by bronchoalveolar lavage from rats infected transtracheally
with Syto 9-labeled bacteria are fluorescently labeled for identification and analyzed by flow cytometry to
quantify their bacterial uptake.
© 2010 Elsevier B.V. All rights reserved.
Recent studies have utilized flow cytometry to measure macro-
phage (MΦ) phagocytosis of microorganisms (Stojkovic et al., 2008;
Luo et al., 2006; Gille et al., 2006; Shida et al. 2006; Utaisincharoen
et al., 2003). However, quantifying alveolar MФ phagocytosis by
flow cytometry is problematic due to their high autofluorescence.
Additionally, MΦ phagocytosis assays performed in vitro with pre-
opsonized or modified (e.g. heat-killed) bacteria to facilitate uptake
(Medeiros et al., 2009; Fitzpatrick et al., 2008; Brown et al., 2007;
Kudo et al., 2004) are limited by not adequately mimicking the
biological environment within the infected lung. We therefore
developed an in vivo assay using live, non-opsonized organisms that
allows examination of uptake within the lungs as well as detection of
underlying host immune defects leading to reduced opsonization and/
or phagocytosis during pulmonary infections. Our assay described
herein has the potential for adaptation to other pathogens, other
animal models, and even in vitro human studies.
Staphylococcus aureus 29213, Streptococcus pneumoniae 6303,
Klebsiella pneumoniae 35657, and Escherichia coli 25922 (American
Type Culture Collection, Rockville, MD, USA) were grown on sheep
blood agar plates (Remel, Lenexa, KS, USA) for 16 hours at 37 °C in 5%
CO2. Organisms suspended in sterile water to an optical density of 1.0 at
540 nm (∼3×108 colony forming units (cfu)/ml) were incubated at
room temperature for 15 min in the dark with 10 nM Syto 9 (Molecular
Probes, Eugene, OR, USA). They were washed twice to remove excess
dye and resuspended in sterile water. A FACSAria cytometer (Becton
Dickinson, San Jose, CA, USA)with a 530/30 band pass filter was utilized
to detect Syto 9 fluorescence. Syto 9 labeled staphylococci effectively,
causing them to fluoresce more intensely than the autofluorescence of
rat alveolar MΦs (Fig. 1A). Syto 9 did not alter the viability of the
bacteria, as shown by comparison of plate counts performed before and
after staining (7×108 cfu/ml vs. 5×108 cfu/ml, respectively). To
demonstrate that our assay could be adapted to measure phagocytosis
of other common bacteria, we used Syto 9 to directly stain S.
pneumoniae, K. pneumoniae, and E. coli, all of which stained more
brightly than rat MΦs (Fig. 1B-D). Several other fluorescent dyes were
evaluated, but failed due to leakage (DAPI and Hoechst 33258), low
fluorescent intensity (CT Blue CMAC and Qdot 800) or the need for
species-specific reagents (APC-Cy7).
Pathogen-free, male Sprague-Dawley rats (Charles River Labora-
tories, Kingston, NY, USA) were infected transtracheally with
1×108 cfu of Syto 9-labeled staphylococci under light anesthesia as
described previously (Vander Top et al., 2006). To prevent the loss of
bacterial fluorescent intensity due to bacterial lysis within the MФs,
the rats were euthanized exactly 15 minutes post-infection by an
intraperitoneal injection of 75 mg/kg body weight of pentobarbital
(Nembutal, Abbott Laboratories, Abbott Park, IL, USA) followed by
exsanguination. Ex vivo bronchoalveolar lavage was performed to
collect pulmonary cells from the rats’ lungs as described previously
(Preheim et al., 1991). The recovered cells were stained with
phycoerythrin (PE)-conjugated antibody (RP-1; BD Pharmingen,
San Diego, CA, USA) to label neutrophils and a biotinylated antibody
Journal of Microbiological Methods 81 (2010) 194–196
⁎ Corresponding author. Department of Microbiology and Immunology, Creighton
University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.
Tel.: +1 402 280 2346; fax: +1 402 280 1875.
E-mail address: mgentry@creighton.edu (M.J. Gentry-Nielsen).
0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2010.02.005
Contents lists available at ScienceDirect
Journal of Microbiological Methods
j ourna l homepage: www.e lsev ie r.com/ locate / jmicmeth
(1C7; BD Pharmingen) followed by streptavidin-allophycocyanin
(APC) (BD Pharmingen) to label MФs (Vander Top et al., 2006).
Three-color flow cytometric analysis was performed using dual
laser excitation (488 nm and 637 nm). On each assay day, MФs
collected from a control rat infected with non-fluorescent staphylo-
cocci were used to exclude autofluorescence in the Syto 9 channel.
Based on scatter characteristics, a minimum of 10,000 cells were
analyzed in the APC (660 nm) channel for MФs and PE (575 nm)
channel for neutrophils. The percentage of MФs with associated
bacteria was determined as the percentage of APC positive cells
fluorescing brighter in the Syto 9 (530 nm) channel than MФs from
the control rat, as analyzed using FlowJo Software (Tree Star, Ashland,
OR, USA).
Confocal microscopy was performed to ensure that staphylococci
in positive MΦs had been internalized rather than bound to the cell
surface. Cytospin slides prepared from lavage samples of each rat
were examined with a scanning confocal microscope (Zeiss Micro-
Imaging, Thornwood, NY, USA). Syto 9-labeled staphylococci were
imaged using 488 nm excitation and a band pass filter of 500-530 nm.
A Chameleon ULTRA titanium:sapphire laser (Coherent, Santa Clara,
CA, USA) was used for two-photon excitation (760 nm) of DAPI-
stained MФ nuclei (435-485 nm band pass filter). Syto 9-labeled
staphylococci were observed throughout MФs that were optically
sectioned at 0.82 μm intervals. Individual MФ images in the same
focal plane as MФ nuclei indicated the presence or absence of
cytoplasmic bacteria as depicted in Figs. 2A and B, respectively. Fewer
than 2% of the positive cells demonstrated 1-2 surface-associated
bacteria, verifying that the uptake measured was from phagocytosed
organisms.
Manual counts of cytospin slides stained with hematoxylin and
eosin also were performed to confirm the MΦ phagocytosis values
determined by flow cytometry. Two hundred cells from each sample
were counted independently by two technicians using a light
microscope to quantify the percentage of MФs containing phagocy-
tosed staphylococci (Fig. 2C) vs. those without internalized bacteria
(Fig. 2D). The flow cytometry and light microscopy phagocytosis
values from 17 rats were compared by regression analysis (Fig. 3). The
mean percent of MΦ phagocytosis determined by flow cytometry was
23.5% and by manual counting was 16.6%, with a Spearman's rank
order correlation coefficient of 0.63 (p=0.007). Manual counts of
MФs from control rats infected with unstained bacteria also were
similar, indicating that Syto 9 did not affect staphylococcal virulence
or adherence.
In conclusion, we have developed a flow cytometric assay to
quantify bacterial uptake by MФs in vivo. The in vivo assay allows the
determination to be made within the lungs of experimental animals
during an actual infection. Although the assay was developed with S.
aureus in rat lungs, successful staining of additional bacterial species
indicates it can be adapted to other microorganisms and alternative in
vivo animal models, as well as to quantification of uptake by human
alveolar MФs in vitro.
The authors would like to thank Mary Snitily for her skilled
technical assistance in development of the assay. This research was
supported by revenue from Nebraska cigarette taxes awarded to
Fig. 1. Syto9-labeledbacteria. S aureus (A), S. pneumoniae (B),K. pneumoniae (C), andE. coli (D)were successfully labeledwith Syto 9andflourescedmore intensely thanMФ autofluorescence.
195A.M. Pitz et al. / Journal of Microbiological Methods 81 (2010) 194–196
Creighton University by the Nebraska Department of Health and
Human Services. It was performed with resources and the use of
facilities at the Omaha Veterans Affairs Medical Center and was
approved by the VA Animal Use and Care Committee. Confocal
microscopy was conducted at the Integrative Biological Imaging
Facility at Creighton University, which is supported by Creighton
University School of Medicine and constructed with support from C06
Grant RR17417-01 from the NCRR, NIH.
References
Brown, L.A., Ping, X.D., Harris, F.L., Gauthier, T.W., 2007. Glutathione availability
modulates alveolar macrophage function in the chronic ethanol-fed rat. Am. J.
Physiol. Lung. Cell. Mol. Physiol. 292, L824–L832.
Fitzpatrick, A.M., Holguin, F., Teague, W.G., Brown, L.A., 2008. Alveolar macrophage
phagocytosis is impaired in children with poorly controlled asthma. J. Allergy. Clin.
Immunol. 121, 1372–1378.
Gille, C., Spring, B., Tewes, L., Poets, C.F., Orlikowsky, T., 2006. A newmethod to quantify
phagocytosis and intracellular degradation using green fluorescent protein-labeled
Escherichia coli: comparison of cord blood macrophages and peripheral blood
macrophages of healthy adults. Cytometry 69, 152–154.
Kudo, K., Sano, H., Takahashi, H., Kuronuma, K., Yokota, S., Fujii, N., Shimada, K., Yano, I.,
Kumazawa, Y., Voelker, D.R., Abe, S., Kuroki, Y., 2004. Pulmonary collectins enhance
phagocytosis of Mycobacterium avium through increased activity of mannose
receptor. J. Immunol. 172, 7592–7602.
Luo, Y., Cook, E., Fries, B.C., Casadevall, A., 2006. Phagocytic efficacy of macrophage-like
cells as a function of cell cycle and Fcgamma receptors (FcgammaR) and
complement receptor (CR)3 expression. Clin. Exp. Immunol. 145, 380–387.
Medeiros, A.I., Serezani, C.H., Lee, S.P., Peters-Golden, M., 2009. Efferocytosis impairs
pulmonary macrophage and lung antibacterial function via PGE2/EP2 signaling.
J. Exp. Med. 206, 61–68.
Preheim, L.C., Gentry, M.J., Snitily, M.U., 1991. Pulmonary recruitment, adherence, and
chemotaxis of neutrophils in a rat model of cirrhosis and pneumococcal
pneumonia. J. Infect. Dis. 164, 1203–1206.
Shida, K., Kiyoshima-Shibata, J., Nagaoka, M., Watanabe, K., Nanno, M., 2006. Induction
of interleukin-12 by Lactobacillus strains having a rigid cell wall resistant to
intracellular digestion. J. Dairy. Sci. 89, 3306–3317.
Stojkovic, B., Torres, E.M., Prouty, A.M., Patel, H.K., Zhuang, L., Koehler, T.M., Ballard, J.D.,
Blanke, S.R., 2008. High-throughput, single-cell analysis of macrophage interac-
tions with fluorescently labeled Bacillus anthracis spores. Appl. Environ. Microbiol.
74, 5201–5210.
Utaisincharoen, P., Kespichayawattana, W., Anuntagool, N., Chaisuriya, P., Pichyangkul,
S., Krieg, A.M., Sirisinha, S., 2003. CpG ODN enhances uptake of bacteria by mouse
macrophages. Clin. Exp. Immunol. 132, 70–75.
Vander Top, E.A., Perry, G.A., Gentry-Nielsen, M.J., 2006. A novel flow cytometric assay for
measurement of in vivo pulmonary neutrophil phagocytosis. BMC. Microbiol. 6, 61.
Fig. 2.Microscopic images ofMФswith andwithout bacteria. A 0.82 µm thick optical slice of aMФ displaysfluorescent puncta of varying sizes indicating the presence of internalized Syto-
9-labeled bacteria in multiple cytoplasmic foci (A) compared to a MФ without bacteria (B). As a comparison to flow cytometry, two hundred cells from each rat were counted by light
microscopy to determine the percentage of MФs that phagocytosed bacteria. A MФ that ingested bacteria (C) was easily identified from aMФ that had no bacteria (D). Scale bar=5 µm.
Fig. 3. Comparison of MФ phagocytosis values as determined by flow cytometry and
light microscopy. A strong correlation exists between both values (p=0.007).
196 A.M. Pitz et al. / Journal of Microbiological Methods 81 (2010) 194–196


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A flow cytometric assay to quantify \u3ci\u3ein vivo\u3c/i\u3e bacterial uptake by alveolar macrophages

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