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Respiratory Bursts in Garlic Treated Macrophages
Shannon Proctor
Departments of Chemistry and Biology
Jacksonville University
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MacrophagesGreek for “big eaters”Originate from white blood cells called monocytesThey play many roles in the bodyMajor role in immunity and the immune response
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Electron micrograph of a macrophage attacking bacteria.
Representation of macrophages that I worked with using an inverted microscope.
www.hartnell.edu/biology
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BackgroundGarlic has been used since ancient times as an
herbal remedy.Heart diseaseTumorsThe PlagueAntibacterial properties
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BackgroundPrevious research has shown that garlic enhances
macrophage activity.Leishmania major, Ghazanfari 2005Augmentation of function, Lau 1991Enhanced immunocompetence, Lamm 2001
Our lab has shown that garlic does increase phagocytosis in macrophages
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Internalization vs. DigestionMacrophages will phagocytose many things
Engulf the particleMay or may not destroy
There is a chemical change when they digestO2 uptake increasesRapid release of reactive oxygen speciesThis is what we want to measure
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AbstractAn increase in the respiratory burst would be
significant
Nitro-blue tetrazolium (NBT)Yellow solution that turns blue
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Cell CultureIC-21 Cells
Cultured in RPMI media
Incubated at 37°C, 5% CO2
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Phagocytosis AssaySubcultured at
100% confluency
DetachedResuspended in control and media with garlic (0.5% or
1%)Incubation for 3hrsAddition of latex beads and fresh media
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Phagocytosis AssayRepeated every 30, 60, and 90 minutesIncubation throughoutAssay stopped by washingStaining using Hema 3 kitInverted microscope for viewing175-250 cells were counted and analyzed
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8-Well Slide
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Results
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Results
Percent phagocytosis measured using the following equation:(# of macrophages with engulfed bead ÷ total # macrophages) X 100
D. Lindsey and K. Jackson, 2006
IC-21 Phagocytosis5 ul/ml Garlic
0.00
5.00
10.00
15.00
20.00
25.00
30 60
Time (minutes)
% o
f to
tal c
ells
p
hag
ocy
tose
*= Control (60 % ethanol)
= Garlic
* P=.055, Student’s T-test
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ResultsNBT Test
Dissolve 1 tablet in waterFix cells (no staining)Cover cells with NBT solution and incubate at 37°C for
30 minutesRinse with filtered PBSObserve color under microscope
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ConclusionFuture research could use latex beads to ensure
phagocytosis and use a bacterium, for example, to measure the respiratory burst
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AcknowledgementsDr. Karen Jackson, advisor
Patricia Roman and Curtis Dobrowolski
Jacksonville University Public Safety
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ReferencesBuescher, E., Alling, D., and Gallin, J. (1985) Use of an x-
linked human neutrophil marker to estimate timing of lyonization and size of the dividing stem cell pool. Journal of Clinical Investigation 76 (4), 1581-1584.
Ghazanfari, T., Hassan, Z., and Khamesipour, A. (2005). Enhancement of peritoneal macrophage phagocytic activity against Leishmania major by garlic (Allium Sativum) treatment. Journal of Ethnopharmacology 103 (3), 333-337.
Lindsey, D. and Jackson, K. (2006). Allium sativum Effects on Phagocytic Activity of IC-21 Peritoneal Macrophages in vitro