Cell Biology Lab Midterm Review
Terms
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- Cell Culture
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Doing things In Vitro (outside the body) to minimize In Vivo conditions
-Cell grows in monolayer(one layer) - How to obtain a cell for In Vitro
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-Chop lung tissue up and use enzymes to break them up
-Dissociated cells in petri dish and put it in something it can grow in
-Enzymes used:
--Trypsin
--Collagenase - Media to help cell grow and its components
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RPMI
-Phenol Red(pH indicator)
-Amino acids
-Glucose
-Vitamins
-Salts
-Bicarbonate - What makes a cell happy?
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Temperature(37C)
Food
pH(from incubator and bicarbonate)
SPACE - Confluency
- physical observation of space to cell ratio (Microscope)
- Hemocytometer and how to use it
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Used to count cells (GRID)
-Look at worksheet to learn how to use it - Lab 4-Cell Viability Probes
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Viability=Living
Probes=Tag - Cell type used
- RAW 264.7 Mouse Cancer cells
- Purpose of Lab 4
- Determine the number of living cells in each well using CalceinAM
- CalceinAM
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Fluorogenic Esterase Substrate
-Passively enters the cell(all cells)
-Diffuses from high concentrations to low concentrations
-Measures ENZYME ACTIVITY and MEMBRANE INTEGRITY
-Made in stock of (1-10mM) in DMSO (we use 1uM) - Enzyme Activity
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If the cell is enzymatically active, esterases will cleave portion of CalceinAM
-Cleaving of CalceinAM gives it a (-) charge
-(-) charge does not allow the Calcein to diffuse out of the membrane (Fluorescence) - CalceinAM reaction to DEAD cells
- if the cell is dead or damaged, CalceinAM will diffuse out (No Fluorescence)
- 2 Things that favor Retention of Calcein
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-Low incubation temperature(37C)
-Highly charged esterase products - DMSO
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Freezing agent
-Allows things to freeze quickly and thaw slowly
-Preserves the molecule when taken out of incubation - Calcein AM Facts II
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-Indicator of Cell Viability
-Has superior retention
-Insensitivity of fluorescence to pH (cell we use pH=7)
-Does not interfere with cell activities
-Has 6(-) and 2(+) charges (overall 4+ charge) - Equipment to show how many cells light up
- CytofluorII - gives Fluorescent Unit(FU) based on number of cells which take up the CalceinAM and cleave the substrate
- Lab 5
- Cell Viability in Response to Hydrogen Peroxide Treatment
- Lab 5 Purpose
- To determine cell viability in response to Hydrogen Peroxide treatment
- Respiration Equation
- C6H12O6 + O2 --> 6CO2 + 6H2O
- ROS
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Type of free radical that can damage the cell membrane, mitochondria, and DNA
-Generated constantly as a part of life
-Formed during enzymatic reactions - Free Radicals
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DANGER
-Produced by UV Light, radiation, smoking, pollution - Good Biological Effects of ROS
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-Needed/essential to life
-Used by phagocytic cells to kill invaders - Bad Biological Effects of ROS
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-Toxic to cells
-Very reactive
-*Damage to cell membranes* - Lipid Peroxidation
- Target unsaturated fatty acids(double bonds)
- Effects of Lipid Peroxidation
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-Altered permeability
-Increased membrane rigidity
-Decreased activity of membrane bound enzymes
-Altered activity of membrane receptors
All these cause the cell to die - Enzymatic Antioxidants
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Protect cells from oxidative stress
-Superoxide Dismutases (SOD)
-Catalase
-Glutathione Peroxidase - Non-Enzymatic Antioxidants
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Things you eat
-VitaminE-soluble in fats(membranes)
--Trap peroxy radicals in membrane
-VitaminC-soluble in water(cytosol)
--Removes peroxy radicals from membrane
-Glutathione-most important intracellular defense
--Tripeptid-contains cystein to provide an exposed SH group
--SH is reactive and acts as alternative target for radicals - Calculate Percent Viability
- (Average RFU)/(Cell number in blank)