Fall 2015Amelia Payne | Big Picture South Burlington

Introduction

• Feel free to use electronic devices during my presentation

• Ask questions during presentation

• You will get candy

Introduction

• Internship

• Classes

• Online Learning

• Time tracking

• Clubs

• STP

Internship At Weiss Lab

Freezing Decellurized Lungs

• Cultivate cells in sterile conditions

• Freeze tissue in different mediums

• Defrost tissues and seed with cells

• Test for cell growth with WST-assay

Growing Cell Lines Human Lung Fibroblasts (HLF)

• Defrosted cells

• Grew under sterile conditions

• Counted

• Replated and frozen

Counting

• Inject 10 µl of cell suspension into “Neubauer chamber” (device for cell counting)

• Count number of cells in one quadrant

• Multiply by 10^4 to get total cells per ml cell suspension

Freezing Lungs• At rate of 1°C per minute

to -80°C

• Transferred to liquid nitrogen (-195°C = -320°F) for long term storage

• Will defrost at 1, 3, 6, and 12 months and do WST-assay

• Control: fresh tissue

• We froze tissue in

• 5% glycerol

• 10 % DMSO in PBS

Seeding Defrosted Tissue

We will defrost tissue rapidly and seed with cells once we reach the first time points.

WST-assay

• Indicates metabolic activity of cells

• WST-1 (water soluble tertrazolium salt-1, red) is cleaved into formazan (yellow) by an enzyme produced by the mitochondria. The resultant color changes indicates metabolic activity of the cells

• Absorbance measurement (440 nm) to determine how much formazan was produces

Data from WST Assay Control

The Team

Thanks to all my awesome mentors who are here today

*Sorry if you’re not in the picture because you weren't at lunch on Thursday.