Experiment 3 Flashcards
(19 cards)
Standard deviation
measurement of how the values vary from the mean
In our experiments what 2 things must you always report
Mean +/- Sd and values are significantly different (if means +/- 1SD do not overlap there is a significant difference between the two values)
What signals can be present for cell division?
hormones, growth factors, chemicals and physical agents
EGF
Small protein growth factor that is typical of several different growth factors.
Produced by a number of cell types
Affects several different cell types
–>affected cells must ahve EGF receptors
–> paracrine= affects cells nearby
–> autocrine affects itself
Induces cells in G0 or G1 phase to enter S phase
Name the techniques in which cell proliferation can be measured (4)
Tritiated thymine uptake, DNA binding dyes, measurement of enzymatic activity and cell counts
Which uses a hemocytomer? Countess II or CyQuant
Countess II
Cyquant
a cell permeant DNa bidning dye will be used to quantify cell numbers, a set of controls with standardized cell numbers will be used so that a calibration curve can be preapred to enable the estimation of cell numbers
characteristics: membrane permeable, colorless, binds to DNA, absorbs light, emits fluoresent light
Procedure: wash cells to remove dead cells, add dye to the culture and incubate, and measure florescence using the fluorometer
Benefits of cyquant
non radioactive, similar to 3H-thumidine incorporation, simple to do, fluorometer can read 96, 24 and 12 well plates
problems of cyquant
potential mutagens, syto dyes are expensive, can measure DNA of dead cells
Countess II
Characteristics: Uses a hemacytometer, uses a camera with an autofocusing feature to take an image of the cells on the hemacytometer. Then the instrument uses counting algorithms to identify and count the cell number cells (live/unstained and dead/stained if stained with trypan blue) in cell sample
Procedure: stain cells with trypan blue, counts in 10 seconds, distinguishes between live and dead cells by: cell size, brightness and cell circularity
Calculates live cells/ml and viability
What technique do you use for cell growth, proliferation or number?
Counting cells with a hemacytometer, syto24, countess II and CyQuant
Should EGF have an effect on the proliferation of the 3T3 cells?
Yes the EGF should have an effect because the 3T3 express EGF receptors which then activates intracellular pathways that promote DNA synthesis and cell cycle progression
Can the Countess II be used to determine the number of apoptotic cells in a culture? Why or why not?
No, Countess II can only determine the alive and dead cells but it cannot determine wether the dead cells are apoptotic or necrotic
What is Grb2 and what is its relation to the activation of the EGF receptor?
Adaptor protein that is invovled in signal transduction, in particular the EGF receptor signaling pathway. It is necessary for downstraeming the signaling pathways by binding to phosphorylated tyrosine on the activated EGF receptor
Define a kinase and including what two things the enzymes target to act upon.
Type of enzyme that transfer a phosphate group from a high energy molecule, ATP to a specific target molecule.
They target proteins and lipids
Two advantages of Countess II compared to a hemacytometer
increase spead of counting cells and reduces human error
Two disadvantages to Countess II
May fail to differentiate between cell and clumps of cells
Cannot determine apopototic vs. necrotic
Two advantages of Countess II compared to Cyquant
Countess II gives live and dead cells, while CyQuant gives total number of cells
Countess II does not require fluorescent dye making it simpler and not requiring fluorescence detection instrument
Two advantages of CyQuant
fluorescent dye binds directly to DNA and is compatable with multi-well plates
