Difference between revisions of "20.109(S17):Complete cell viability assay (Day9)"

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(Part 1: Evaluate cell viability)
Line 11: Line 11:
  
 
#Retrieve your plate from the 37 °C incubator.
 
#Retrieve your plate from the 37 °C incubator.
 +
#Briefly look at the DLD1 and BRCA2-/- cells under the microscope.
 +
#*Make a note of their confluency and morphology.
 
#Aspirate the spent media from each well.
 
#Aspirate the spent media from each well.
 
#*Be careful not to cross-contaminate between the wells.
 
#*Be careful not to cross-contaminate between the wells.

Revision as of 01:46, 13 April 2018

20.109(S18): Laboratory Fundamentals of Biological Engineering

Sp18 banner image v2.png

Spring 2018 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Assessing ligand binding        2. Measuring gene expression        3. Engineering biomaterials              


Introduction

The CellTiter-Glo Luminescent Cell Viability Assay is a method for quantifying the number of viable cells based on measuring the amount of ATP present. ATP is a proxy for the presence of metabolically active (alive) cells. In this assay, the cells are lysed and ATP is released from the active cells. In a reaction catalyzed by a propriety luciferase enzyme, luciferin, ATP, and oxygen result in oxyluciferin, AMP, PPi, carbon dioxide, and light. The light product is then measured using a luminometer.

Protocols

Part 1: Evaluate cell viability

Today you will complete the second experiment for Mod 2 - the cell viability assay. In the previous class you used etoposide to induce DNA damage and added a NHEJ inhibitor. By comparing the results of these treatments between the DLD-1 and BRCA2- cell lines you will more deeply examine the DNA repair pathways from lecture.

  1. Retrieve your plate from the 37 °C incubator.
  2. Briefly look at the DLD1 and BRCA2-/- cells under the microscope.
    • Make a note of their confluency and morphology.
  3. Aspirate the spent media from each well.
    • Be careful not to cross-contaminate between the wells.
  4. Add 500 μL of fresh media into each well.
  5. Obtain an aliquot of the CellTiter Glo reagent from the front laboratory bench.
  6. Thoroughly mix the CellTiter Glo reagent, then add 500 μL into each well of your plate.
    • After adding the reagent, pipet up and down ten times to mix.
  7. Move your plate to the plate shaker at the front laboratory bench and shake for 2 min.
  8. Remove your plate from the plate shaker and incubate on your benchtop for 10 min.
  9. Transfer 100 μL from each well into a white 96-well plate.
    • Be very careful that you add your samples to the appropriate wells according the plate map below.
    • Instructors will add media only control to the 96-well plate to obtain a value for background luminescence.
  10. When all teams have transferred their samples, the teaching faculty will take measure the luminescence using a plate reader in the BMC and upload the data to the Class data page.
Sp18 20.109 CTG plate map.png

Part 2: Complete data analysis

Pool the data by drug and use the statistical tools you learned in Mod1 to analyze the relevant class data for your M2 Research Article.

Reagents

  • CellTiter Glo cell viability assay kit (Promega)

Navigation links

Next day: Grow phage-based active (cathode) material

Previous day: Induce DNA damage and apply drug treatments for cell viability and identification of regulatory motifs in RNA-seq data