Research Tools and Resources - RIC...
Transcript of Research Tools and Resources - RIC...
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Research Tools and Resources!or!
Swiss Army Knives !and a Peanut Butter Sandwich
Microbiology & Molecular Biology Department College of Life Science Brigham Young University
Research Tools and Resources
• Why worry about tools?
• What tools are available?
• What financial resources are available?
• Do you have the right piece of equipment?
• Do you have the experience or is an experienced person able to help you?
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College of Life Sciences!Core Facilities
• Research Instrumentation Core • Microscopy (confocal & EM) • DNA Sequencing Center • Chromatography • Soil and Plant Analysis
• Procure Equipment
• House and Maintain Equipment
• Educate
Imaging gels & screens
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Absorbance, Fluorescence, Luminescence
Centrifugation
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Microscopy
Flow Cytometry
Flow Cytometry
LASER Forward Scatter Detector
(size)
Side Scatter Detector (granularity)
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Flow Cytometry
LASER Specific Antibody
with Fluorescent Tag
(protein expression)
Red Fluorescence Detector
Flow Cytometry
LASER
Flow Cytometry
LASER Forward Scatter Detector
Side Scatter Detector
Green Detector
Blue Detector
Red Detector
(size)
(granularity)
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Flow Cytometry
LASER Forward Scatter Detector
Side Scatter Detector
Green Detector
Blue Detector
Red Detector
Mirrors
(size)
(granularity)
Flow!Cytometry! Data
CD11C
CD8a
B220
GR1
Mac1
GFP
Laser to PMT’s
+ -
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FACS Data A B C
D E
G
F
• Procure Equipment
o Equipment recommendations for purchase
• House and Maintain Equipment
• Educate
House and Maintain Equipment
873 WIDB
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• Use the tool properly
• Experimental Controls
• Instrumentation Controls
Experimental Controls
I.P. Drug Treatment Transgenic Mouse
Observation: I.P. Drug Treatment into a transgenic mouse for macrophage depletion results spontaneous peritoneal adhesion formation.
Hypothesis: Macrophage depletion results in spontaneous peritoneal adhesion formation.
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Experimental Controls
I.P. Drug Treatment Transgenic Mouse
Observation: I.P. Drug Treatment into a transgenic mouse for macrophage depletion results spontaneous peritoneal adhesion formation.
Hypothesis: Macrophage depletion results in spontaneous peritoneal adhesion formation.
How do I test the hypothesis? What parameters do I consider?
1. Test Subjects
Strain of mouse Gender Age Housing conditions Food Light cycles Room temperature
Experimental Controls
I.P. Drug Treatment Transgenic Mouse
Observation: I.P. Drug Treatment into a transgenic mouse for macrophage depletion results spontaneous peritoneal adhesion formation.
Hypothesis: Macrophage depletion results in spontaneous peritoneal adhesion formation.
How do I test the hypothesis? What parameters do I consider?
1. Test Subjects
2. Treatments
Diluent for drug Drug concentration Route of administration Volume to deliver Mock treatments
Experimental Controls
I.P. Drug Treatment Transgenic Mouse
Observation: I.P. Drug Treatment into a transgenic mouse for macrophage depletion results spontaneous peritoneal adhesion formation.
Hypothesis: Macrophage depletion results in spontaneous peritoneal adhesion formation.
How do I test the hypothesis? What parameters do I consider?
1. Test Subjects
3. Data Collection 2. Treatments
When to collect data What samples to take How to take samples What method of euthanasia How to process samples What equipment for sample analysis What type of data is generated What controls are needed
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Experimental Controls
I.P. Drug Treatment Transgenic Mouse
Observation: I.P. Drug Treatment into a transgenic mouse for macrophage depletion results spontaneous peritoneal adhesion formation.
Hypothesis: Macrophage depletion results in spontaneous peritoneal adhesion formation.
How do I test the hypothesis? What parameters do I consider?
1. Test Subjects
4. Data Interpretation
3. Data Collection 2. Treatments
How to interpret readings Which samples will be compared Is the data numerical Is the data quantitative Is the data qualitative What statistics will be used
Will the data yield applicable information to support or refute the hypothesis
Experimental Controls
I.P. Drug Treatment Transgenic Mouse
I.V. Drug Treatment Transgenic Mouse
I.P. Mock injection (diluent) Transgenic Mouse
I.P. Drug Treatment Wildtype mouse
Observation: I.P. Drug Treatment into a transgenic mouse for macrophage depletion results spontaneous peritoneal adhesion formation.
Hypothesis: Macrophage depletion results in spontaneous peritoneal adhesion formation.
S.H. Burnett, B.J. Beus, R. Avdiushko, J. Qualls, A.M. Kaplan and D.A. Cohen (2006) Development of peritoneal adhesions in macrophage depleted mice. J. Surgical Res. 131, 296-301.
Instrumentation Controls Flow Cytometry: Fluorescence Intensity
LASER
Filter for Green Fluorescence
10 1 10 2 10 3 10 4 55
111 167 223
Cou
nts
R1
10 0 0 Green Fluorescence
Positive Control Sample with proper voltage settings on
the PMT detecting green PMT
General light
scatter
Any unstained cells In the sample are measured here
Any cells stained with green-labelled antibody are measured here
The PhotoMultiplier Tube (PMT) measures any light that reaches it and reports the measurement in
intensity on a log scale.
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Instrumentation Controls Flow Cytometry: Fluorescence Intensity
LASER
Filter for Green Fluorescence
10 1 10 2 10 3 10 4 55
111 167 223
Cou
nts
R1
10 0 0 Green Fluorescence
Negative Control Sample with proper voltage settings on the PMT detecting green
PMT
General light
scatter
General light scatter peak should be here
Instrumentation Controls Flow Cytometry: Fluorescence Intensity
LASER
Filter for Green Fluorescence
Voltage controls
sensitivity
10 1 10 2 10 3 10 4 55
111 167 223
Cou
nts
R1
10 0 0 Green Fluorescence
Negative Control Sample with voltage set too high
on PMT for green
Sample should peak here
PMT
General light
scatter
Instrumentation Controls Flow Cytometry: Fluorescence Intensity
LASER
Filter for Green Fluorescence
Voltage controls
sensitivity
10 1 10 2 10 3 10 4 55
111 167 223
Cou
nts
R1
10 0 0 Green Fluorescence
Negative Control Sample with voltage set extremely high
on PMT for green
Sample should peak here
PMT
General light
scatter
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Instrumentation Controls Flow Cytometry:
Fluorescence Overlap
LASER
Filter for Green Fluorescence
10 1 10 2 10 3 10 4 55
111 167 223
Cou
nts
R1
10 0 0 Green Fluorescence
Red Control Sample (reading in green should be negative) Ideally, all red is blocked by the green
filter so that colors can be distinguished PMT
Instrumentation Controls Flow Cytometry:
Fluorescence Overlap
LASER
Filter for Green Fluorescence
10 1 10 2 10 3 10 4 55
111 167 223
Cou
nts
R1
10 0 0 Green Fluorescence
Red Control Sample (reading in green should be negative) Some red fluorescence is able to
cross through the green filter PMT
A small amount of red can pass through the green filter
Instrumentation Controls Flow Cytometry:
Fluorescence Overlap
LASER
Filter for Green Fluorescence
10 1 10 2 10 3 10 4 55
111 167 223
Cou
nts
R1
10 0 0 Green Fluorescence
Red Control Sample (reading in green should be negative) Instrument controls compensate for
“fluorescence overlap” of red PMT
A small amount of red can pass through the green filter
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Instrumentation Controls
Cou
nts
10 0 10 1 10 2 10 3 10 4 0 250 500 750
1000 R1
R1
10 0 10 1 10 2 10 3 10 4 0 43 87
131 175
Cou
nts
10 0 10 1 10 2 10 3 10 4 0 31 62 93
125
Cou
nts R1
10 0 10 1 10 2 10 3 10 4 0 175 350 525 700
Cou
nts R1 R2
10 0 10 1 10 2 10 3 10 4 0 31 62 93
125
Cou
nts R1
• Isotype controls 1 for each color
• Single color controls 1 for each color
This 6-color experiment requires 12 control samples per tissue type
CD11C
CD8a
B220
GR1
Mac1
GFP
• Use the tool properly
• Design your experiment with appropriate controls
• Seek help: - Consultation
- Protocols - Website
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• Do you have the right piece of equipment?
• Do you have the experience or is an experienced person able to help you?
• Are you using the equipment properly?
• Is your experiment designed correctly and does it include appropriate controls (experimental & instrumentation controls)?
• College core facilities (consultations)
• Professors
• Classes (lecture & lab)
• Equipment
• Experimental Design
Peanut Butter Sandwich
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Department ORCA: Office of Research and Creative Activities Graduate School:
Graduate student scholarships Graduate Research Fellowship Award Research Presentation Award Graduate Mentoring Award
Government Entities: National Science Foundation (NSF) Graduate Research Fellowship National Defence Science and Engineering Graduate Fellowship American Association of University Women Selected Profession Fellowship and International Fellowships National Institute of Health (NIH) Graduate Research Fellowship
• RIC Facility Technicians: • Nels Nielson • Ross Ahrendes -Kindal Debenham (undergraduate assistant)
• Undergraduate Students (Mentored Research)
• Collaborators from: BYU, Univ. of KY, Cleveland Clinic/Case Western, Eastern Virginia Med. School
• Department of Microbiology & Molecular Biology
• College of Life Sciences Dean’s Office
Thank you!