Dates

Lecture

Laboratory Session

Return Day

1/17

Introduction to equipment, supplies and aseptic technique.

Presentation 1

Practice handling new equipment and supplies.

Aseptically prepare media in biosafety hood.

Video: In Vitro Insights

Nell lab 1 ppt

Nell rd 1 ppt

lab report info

TBA Keeping a laboratory notebook and preview of report writing

example notebook page

TBA Hemacytometer boot camp

hemacytometer exercise

ppt images of hemacytometer

extra hints for counting on hemacytometer

1/24

Theory and terminology for subculturing of cell line maintenance in vitro

Presentation 2a

Presentation 2b

Subculturing 3T3-L1 fibroblasts using trypsin, inverted phase microscopes, trypan blue exclusion dye, hemacytometers, incubators and ancillary equipment.

Video: In Vitro Insights Module 2

Nell lab 2 ppt

Subculture 3T3-L1 cells for next laboratory session

scale bars

40x tif

20xtif

10xtif

4xtif

1/31

Cell adhesion and interaction with environment (in vivo and in vitro).

Structural aspects of extracellular matrix and its effects on cellular differentiation.

Behavior of transformed cells.

Theory of spectrophotometry

Presentation 3

3T3-L1 adhesion exercise with quantitative WST-1 assay using plate reader.

Subculture 3T3-L1s for actin stain and adipocyte differentiation.

Coat coverslips with fibronectin.

Nell lab 3 ppt

Seed cells onto coverslips, Subculture for future labs.

TAs will fix cells on coverslips 24-48hrs later

Seed well plate of 3T3s for adipocyte differentiation

2/7

Cell cytoskeleton in cellular differentiation and interaction with environment.

Theory and use of fluorescent dyes and microscope.

Actin as a polymer in material science and engineering

Presentation 4

Actin and the ECM

cartilage scaffold

2/11-2/12

Actin stain with rhodamine phalloidin.

Add differentiation cocktail to 3T3s.

Subculture for future labs.

Molecular Probes Online tutorial: fluorescence1 and light sources and filters

Nell lab 4

Start new well plate of 3T3s for differentiation for lysate.

Subculture for future labs

(TA changes adipocyte media)

2/14

Stem cell terminology and use in tissue engineering.

Fibroblasts as stem cells

Introduction to modes of cellular differentiation.

Working with new muscle cell line, serum and antibiotics.

Presentation 5

for today

Stain differentiated adipocytes with Oil Red O.

Receive and manipulate E63 cell line (myoblasts).

Start new adipocyte differentiation.

Online Broadcast: Nova Science Now: Stem Cells (15 min)

Nell lab 5

Prepare myoblast plate (3 day), and feed myotube plate (6 day)

Keep 3T3 cell line as control.

Expand cells for freezing.

2/21

Preview methods of protein analysis to monitor cellular differentiation.

Theory and method of freezing cell lines.

Centrifugation

Creating a lysate using sonication.

Presentation 6

Prepare E63 3 day and 6 day lysates.

Prepare lysate of adipocytes and undifferentiated fibroblasts.

Freeze 3T3 cell line.

Sonication calculation

Nell/Stephen lab 6

No Return Day

2/28

Protein separation by SDS-PAGE theory and practice

Theory of Western Blotting

Understanding roles of myosin and adipsin in differentiated cells.

Presentation 7

Run muscle, adipose and fibroblast lysates on SDS-PAGE gel.

Western Blotting

Analysis of protein molecular weights

Analyze migration time, distance wrt gel density and voltage.

Nell lab 7

No Return Day

3/6

Antigen detection of MHC, adipsin on western blot

Antibodies and antibody production

Transfection as mechanism to force cellular differentiation

Transfection methods

Use of serum free media in industry

Presentation 8a

Presentation 8b

Western Probing for MHC and Adipsin

Working with DNA.

Use FuGENE 6 to transfect CHO cells with GFP-tublulin plasmid construct

Video: Growing Spare Parts

Nell lab 8

Visualize overexpressed tubulin on fluorescent microscope. Quantitation of percent observed transfection.

3/13

Bone as a tissue.

Bone and Material Science/tissue engineering

Thawing cells

Differentiation of D1 ORL UVA mesenchymal stem cells to bone.

Presentation 9a

9b, 9c

Thaw 3T3s previously frozen

Begin differentiation of D1 ORL UVA cell line to become bone

Online video: Scientific American Frontiers:Body Building

trypsinized D1 image 20X (tif)

Nell lab 9

Split D1 stock plate

Change differentiation media

Observe and manipulate thawed 3T3 cells to create stock dish for feeder plate

3/27

Structure of hair follicle.

Primary culture of epithelial cells

Concept of feeder layer

Biosafety levels in laboratories

Presentation 10

Primary culture of student’s own outer root sheath cells on fibroblast feeder plates

Continue differentiation of D1 ORL UVAs

Nell lab 10

Split D1 stock plate

Change D1 differentiation media

Change media on ORS cells

4/3

Introduction to neurons

Classification of cell renewal

Protein microstamping to guide neuron growth

Presentation 11

View ORS colonies on feeder plate, quantitate growth

Stamp coverslips with poly-d-lysine and add neurons

Continue differentiation of D1ORL UVAs

Video: Rebuilding the Brain: Adult Brain Stem Cells

Nell lab 11

Split D1 stock plate

Change D1 differentiation media.

Replace half of neuron media.

4/10

Bone formation and mineralization.

Concepts of bone differentiation analysis

Handling hazardous chemicals

Observing bone differentiation using Von Kossa staining

Presentation 12

Von Kossa stain of four stages of differentiated osteoblasts (undifferentiated, 1-3 week differentiation), visualizing alkaline phosphatase production.

Creation of lysates for next week’s quantitative analysis of bone differentiation

Nell lab 12

No Return Day

4/17

Methods to quantitate bone growth and mineralization, specifically looking at calcium and alkaline phosphatase

Presentation 13

Perform spectrophotometric assays to quantitate calcium and alkaline phosphatase formation.

Cell migration

No Return Day

4/24

Review, complete evaluation forms

Final Exam--comprehensive

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