1. Biomedical Research 2013
Rowan Daiksel
Dr. Torres-Muñoz

2. Experimental Design
Scientific Background
Hypothesis
Aims (Primary and Secondary)
Materials
Methods (Protocols)
Expected Results
Results
Statistical Analyses
Reports:

3. Aims
• Become familiar with cancer research
• To learn about Circulating Tumor Cells (CTC’s)
• Methods to analyze them:
• Microfiltration
• Immunofluorescence (IF)
• Antigen determinants that identify them as CTC
• Quantitation of antigen determinant in CTC

4. Aims
• Become familiar with experimental design
• Controls :
• Biological
• Experimental
• Learn how to culture cancer cells
• Thawing
• Culturing
• Splitting
• Freezing

5. Thawing

6. Cell Culturing
After 24 hours After 48 hours
After 72
hours

8. Cell Splitting
Split Cells into two
25 mL flasks

9. Cell Freezing

10. Fixing for Immunofluorescence
Other Names for
Formaldehyde:
IUPAC Name: Methanal
Methyl aldehyde
Methyl glycol
Methylene oxide
Formalin
Formol

11. Immunofluorescence (IF)
Secondary Anti-Rabbit
Antibody (raised in
goat)
Anti-Cytokeratin
(rabbit)
Blocked with goat
serum

12. Results from
Immunofluorescence
Negative ControlWith Primary and
Secondary Ab

13. Cytokeratin DAPI
Both CK & DAPI

14. Double Staining IF
Anti- CK Ab
(Rabbit)
Secondary
Anti-Rabbit
Ab (goat)
Anti-CD44
MAb
(mouse)
Anti-mouse
Ab (goat)
Blocked with
Goat Serum

15. IF: A431 CK-A594/CD44-A488/DAPI
C. CD44-A488/DAPI
CK-A594/ CD44-A488/DAPI CK-A594/ DAPI
DAPI

16. Double Staining Quantitation
A431 DAPI
CD44 A488A431 CK A595
A431 CK A594 CD44 A488 DAPI Merge

17. Laser Capture Micro-dissection
(LCM)

18. Thank You
Dr. Torres-Muñoz
Dr. Cote
Dr. Datar