Respiratory Bursts in Garlic Treated Macrophages
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Transcript of Respiratory Bursts in Garlic Treated Macrophages
Respiratory Bursts in Garlic Treated Macrophages
Shannon Proctor
Departments of Chemistry and Biology
Jacksonville University
MacrophagesGreek for “big eaters”Originate from white blood cells called monocytesThey play many roles in the bodyMajor role in immunity and the immune response
Electron micrograph of a macrophage attacking bacteria.
Representation of macrophages that I worked with using an inverted microscope.
www.hartnell.edu/biology
BackgroundGarlic has been used since ancient times as an
herbal remedy.Heart diseaseTumorsThe PlagueAntibacterial properties
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
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
AbstractAn increase in the respiratory burst would be
significant
Nitro-blue tetrazolium (NBT)Yellow solution that turns blue
Cell CultureIC-21 Cells
Cultured in RPMI media
Incubated at 37°C, 5% CO2
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
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
8-Well Slide
Results
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
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30 60
Time (minutes)
% o
f to
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*= Control (60 % ethanol)
= Garlic
* P=.055, Student’s T-test
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
ConclusionFuture research could use latex beads to ensure
phagocytosis and use a bacterium, for example, to measure the respiratory burst
AcknowledgementsDr. Karen Jackson, advisor
Patricia Roman and Curtis Dobrowolski
Jacksonville University Public Safety
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