Transcript Respiratory Bursts in Garlic Treated Macrophages

Shannon Proctor
Departments of Chemistry and Biology
Jacksonville University
Macrophages
 Greek for “big eaters”
 Originate from white blood cells called monocytes
 They play many roles in the body
 Major role in immunity and the immune response
Electron micrograph of a macrophage
attacking bacteria.
www.hartnell.edu/biology
Representation of macrophages
that I worked with using an
inverted microscope.
Background
 Garlic has been used since ancient times as an
herbal remedy.
 Heart disease
 Tumors
 The Plague
 Antibacterial properties
Background
 Previous research has shown that garlic enhances
macrophage activity.
 Leishmania major, Ghazanfari 2005
 Augmentation of function, Lau 1991
 Enhanced immunocompetence, Lamm 2001
 Our lab has shown that garlic does increase
phagocytosis in macrophages
Internalization vs. Digestion
 Macrophages will phagocytose many things
 Engulf the particle
 May or may not destroy
 There is a chemical change when they digest
 O2 uptake increases
 Rapid release of reactive oxygen species
 This is what we want to measure
Abstract
 An increase in the respiratory burst would be
significant
 Nitro-blue tetrazolium (NBT)
 Yellow solution that turns blue
Cell Culture
 IC-21 Cells
 Cultured in RPMI media
 Incubated at 37°C, 5% CO2
Phagocytosis Assay
 Subcultured at
100% confluency
 Detached
 Resuspended in control and media with garlic (0.5% or
1%)
 Incubation for 3hrs
 Addition of latex beads and fresh media
Phagocytosis Assay
 Repeated every 30, 60, and 90 minutes
 Incubation throughout
 Assay stopped by washing
 Staining using Hema 3 kit
 Inverted microscope for viewing
 175-250 cells were counted and analyzed
8-Well Slide
Results
Results
IC-21 Phagocytosis
5 ul/ml Garlic
% of total cells
phagocytose
25.00
*
20.00
= Control (60 % ethanol)
15.00
= Garlic
10.00
* P=.055, Student’s T-test
5.00
0.00
30
60
Tim e (m inutes)
Percent phagocytosis measured using the following equation:
(# of macrophages with engulfed bead ÷ total # macrophages) X 100
D. Lindsey and K. Jackson, 2006
Results
 NBT Test
 Dissolve 1 tablet in water
 Fix cells (no staining)
 Cover cells with NBT solution and incubate at 37°C for
30 minutes
 Rinse with filtered PBS
 Observe color under microscope
Conclusion
 Future research could use latex beads to ensure
phagocytosis and use a bacterium, for example, to
measure the respiratory burst
Acknowledgements
 Dr. Karen Jackson, advisor
 Patricia Roman and Curtis Dobrowolski
 Jacksonville University Public Safety
References
 Buescher, 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