BE115+-+Lab04 - Lab 4: Analysis of 3D Cell Cultures 1...

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Page 1 of 6 Bioengineering 115: Cell Biology for Engineers Laboratory 4: Analysis of 3D Cultures Last updated: January 21, 2011 Lab 4: Analysis of 3D Cell Cultures 1 Introduction In this laboratory you will analyze one well from each cell type and each culture type. The analysis will include observation of cell shape, distribution, and number. In addition, you will prepare for the next several weeks 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 and in agarose. To prepare culture extracts suitable for protein quantification and gel electrophoresis. 3 References and Links Live/Dead assay (Invitrogen L3224) 4 Reagents, Supplies & Equipment 4.1 Reagents 1. 2X lysis buffer 100 mM Tris-HCl 1.58g Tris-HCl 300 mM NaCl 1.75g NaCl 10 mM EDTA 0.41g EDTA 2.0% Triton-X 100 2 mL Triton-X 100 pH 7.5 = 100mL 2X LB 2. Phosphate buffered saline (PBS) 3. Live/Dead assay reagent 4. 2X Trypsin 4.2 Supplies 1. Syringes and needles
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Page 2 of 6 Bioengineering 115: Cell Biology for Engineers Laboratory 4: Analysis of 3D Cultures Last updated: January 21, 2011 2. Non-sterile pipets (various sizes) 3. 15 ml centrifuge tubes 4. Ice 5. Scalpel 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 4: Analysis of 3D Cultures
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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+-+Lab04 - Lab 4: Analysis of 3D Cell Cultures 1...

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