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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!