BIOE 298 Sections AB1 and AB2

1. BIOE 298 Sections AB1 and AB2 Lab 6: Freezing of 3T3-L1 Cells and Making Lysates of Four Cell Types February 27-28, 2006

2. Lab #6 Overview Online Quiz Results Pre-lab Exercise Results Review Lab Protocol Final Reminders Complete Lab Exercises Clean Up!

3. Online Quiz Results Stocks of cells are frozen to protect this valuable resource for future use, as cells in continuous culture are prone to - variation (finite cell lines may become senescent, continuous cell lines are prone to genetic instability) - contamination (by microorganisms or cross contamination from other cell lines) - incubator failure - excessive use of time and materials when not in immediate use - need for distribution to other labs Cells should be frozen - slowly - with DMSO in a higher concentration of serum Senescent - EDTA = binds to calcium ions that would normally inhibit trypsinSenescent - EDTA = binds to calcium ions that would normally inhibit trypsin

4. Online Quiz Results Tubes must be balanced when using a centrifuge KEEP EVERYTHING COLD to slow protein destruction during lysate production Lysosomes release enzymes that will destroy proteins during lysate production The PC-12 cell line is derived from the adrenal gland but takes on a neuronal phenotype when cultured on a collagen coated surface The HeLa cell line was the first human aneuploid cell line manipulated in continuous culture Leibovitz L-15 media is best suited for supporting cell growth in non-CO2 equilibrated environments RPMI media supports a wide variety of suspension cultures aneuploid – having an abnormal number of chromosomes (not diploid or haploid)aneuploid – having an abnormal number of chromosomes (not diploid or haploid)

5. Pre-Lab Exercise Results We are using a microtip sonicator Use figure 5 from “Application of Ultrasonic Processors” to create a standard curve Finding setting of 5 for double amplitude of 6?mm

6. Part A: Freezing 3T3-L1 cells Select 70-80% confluent dish from “freezing” and “stock” 10cm dishes from return day last week Rinse with PBS and trysinize as usual Resuspend in 10 mL regular 3T3-L1 media and transfer suspension to conical tube Count cells and calculate volume of freezing media you will need to have 1x106-1x107 viable cells/ml Centrifuge at setting 5 for 5 minutes Remove regular media by pipetting into waste bottle Resuspend cells in calculated volume of freezing media Make up to 2 cryovials of cells to freeze and place them in styrofoam rack in refrigerator Remove used pipet tips and dump out liquid waste when cleaning hood after you complete exercise Groups 6, 1, and 2 will start with this exercise

7. Part B: Lysing Cells by “Instant SDS Sample Buffer Method” Make lysate for fibroblasts, adipocytes, myoblasts, and myotubes this week - “3 Day E63” plate = myoblasts - “7 Day E63” plate = myotubes One partner makes fibroblast and adipocyte lysate, other partner makes myoblast and myotube lysate Observe all cell dishes and well plates and note observations in lab notebook prior to making lysate Take picture of myotubes for lab report #2 Work quickly (but carefully) when making lysate ALWAYS KEEP EVERYTHING COLD!!! Groups 3, 4, and 5 will start with this exercise

8. Part B: Lysing Cells by “Instant SDS Sample Buffer Method” Basic Procedure: - wash plate 2x with 1ml cold serum free media (found in refrigerator) - wash plate 2x with 1ml cold non-sterile PBS (found in refrigerator, put it back when finished) - add chilled sample buffer to plate (aliquot found in fume hood, put it back when finished) - scrape cells off plate and pipet into 1.5ml eppendorf tubes - use sonicator to pulse cells 10 sec 3x at setting #5 - spin cells in microfuge for 1min at setting #2 - vortex and spin briefly in minifuge - aliquot into 50 µl samples in 0.5ml eppendorf tubes (no more than 3 aliquots per lysate sample) - use prewritten labels and add group & section # - put aliquots in lysate box for your section in -200C freezer

9. Part B: Lysing Cells by “Instant SDS Sample Buffer Method” Additional Notes: - keep all tubes, plates, scrapers, and solubtions on ice block or in ice bucket or in ice cup at all times!! - use one scraper for fibroblasts and adipocytes and one scraper for myoblasts and myotubes - 2X sample buffer is thick, runny and smelly - exchange ice cups often (there are extra in the freezer) and leave unused ice cups in freezer - share ice buckets between groups Clean Up!! - bleach extra lysate that you do not aliquot - bleach lysate well plates and 6cm dishes - throw away pipettes, used pipet tips, and scrapers - rinse waste containers - dump liquid waste and rinse out bottle

10. Final Reminders Email Lab #5 Post Lab Exercise results to Nell - deadline is Thursday March 2nd at 5pm Lab Report #1 (covering Adhesion and Actin Staining Labs) - due Tuesday March 7th at 5pm - come to office hours Friday 1-3pm in Beckman 4159 if you have questions or want to look at the textbook All unused 3T3-L1 stock dishes should be bleached and removed from incubator tonight – you should have nothing remaining in the incubator after this lab Any extra E63 media should be poured out No return day this week – work on your lab report