BE115+-+Lab03 - Lab 3: Analysis of 2D Cultures 1...

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Page 1 of 6 Bioengineering 115: Cell Biology for Engineers Laboratory : Analysis of 2D Cultures Last updated: January 21, 2011 Lab 3: Analysis of 2D Cultures 1 Introduction In this laboratory you will analyze one your 2D cultures from last week. The analysis will include observation of cell shape, distribution, and number. In addition, you will prepare for future labs by extracting your remaining cultures for protein analysis. This means that you will use a lysis buffer (along with some mechanical force) to break the cells open and bring the proteins into solution. For these experiments you and your partner will perform all steps together. Since the cells will not be cultured further, sterile technique will not be necessary. Therefore, all work can be performed at your lab bench, except where microscopy is required. 2 Objectives To observe and measure cell shape and density differences between Rex and 3T3 cells cultured on tissue culture plastic. To prepare culture extracts suitable for protein quantification and gel electrophoresis. 3 References and Links Actin staining (http://www.invitrogen.com/site/us/en/home/References/Molecular- Probes-The-Handbook/Probes-for-Cytoskeletal-Proteins/Probes-for- Actin.html#head2) 4 Reagents, Supplies & Equipment 4.1 Reagents 1. 1X lysis buffer 50 mM Tris-HCl 150 mM NaCl 5 mM EDTA 1.0% Triton-X 100 pH 7.5 2. Phosphate buffered saline (PBS) 3. 1X Trypsin/EDTA 4. Trypan blue 5. 1.0% glutaraldehyde in PBS
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Page 2 of 6 Bioengineering 115: Cell Biology for Engineers Laboratory : Analysis of 2D Cultures Last updated: January 21, 2011 6. 0.5% Triton-X 100 in PBS 7. Rhodamine phalloidin (Invitrogen R415 - a 1:200 dilution of the original solution in PBS) 8. ProLong® Gold antifade reagent with DAPI (Invitrogen P-36931) 4.2 Supplies 1. Cell scrapers 2. Syringes and needles 3. Non-sterile pipets (various sizes) 4. Pasteur pipettes 5. 15 ml centrifuge tubes 6. 50 ml centrifuge tubes 7. Ice 8. Scalpel 9. 1.5 ml centrifuge tubes 10. Kimwipes 11. Glass slides 4.3 Equipment 1. Inverted microscope 2. Fluorescence microscope 3. Hemacytometer
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Page 3 of 6 Bioengineering 115: Cell Biology for Engineers Laboratory : Analysis of 2D Cultures Last updated: January 21, 2011 5 Protocols Everyone should begin by removing their 2D cell cultures from last week and observing
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This note was uploaded on 10/14/2011 for the course BIOE 115 taught by Professor Haley during the Fall '11 term at City College of San Francisco.

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BE115+-+Lab03 - Lab 3: Analysis of 2D Cultures 1...

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