Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
150 Cards in this Set
- Front
- Back
|
What are the non-adherent cells of the blood? What are their colors?
|
Erythrocytes are red and Leukocytes are white.
|
|
|
What does it mean when the number of neutrophils is high?
|
Infections
|
|
|
What does it mean when the number of eosnophils is high?
|
parasite infections
|
|
|
What happens to the T helper cells when an individual has AIDS?
|
The number of T helper cells drops
|
|
|
What do we label antibodies with?
|
Enzymes, these could be horseradish, AP
|
|
|
The spleen cell cultures, How did the control and the stimulated spleen cells look?
|
The stimulated cells looked clumped and the control looked normal. The stimulated clumped because LPS makes them move from G0 into the cell cycle
|
|
|
How do we identify proteins using SDS-PAGE?
|
By molecular weight
|
|
|
How do we identify proteins using Western Blotting?
|
By specific antibodies
|
|
|
How do we identify proteins using Immunofluorescent staining?
|
By specific antibodies
|
|
|
What can we do to quantify the amount of protein we have in solution?
|
We can scan or western blot the bands to compare band densities. We can also use specific assays for particular proteins and their activities. But both of the above are limited
|
|
|
When referring to a good assay, what is specifity?
|
measure only the specific protein in a complex mixture (serum, culture, supernatant, etc)
|
|
|
When referring to a good assay, what is sensitivity?
|
the ability to measure small quantities of the specific protein (micro, nano, pico)
|
|
|
For an assay to be a good one, why does it have to be simple?
|
the more steps you take in an experiment the more you are removed form your original sample. Also, you have to be able to do many sample and not being simple complicates this.
|
|
|
What does ELISA stand for?
|
Enzyme Linked Immunosorbent Assay
|
|
|
How does ELISA work?
|
It uses specific antibodies to recognize and quantitate a specific protein even in a complex mixture. It can be used for anything you can make an antibody for.
|
|
|
Why does ELISA use enzyme conjugated antibodies?
|
to give it high amplification and high sensitivity
|
|
|
What is the difference between specificity and sensitivity?
|
specificity is the ability to measures only a specific protein, while sensitivity is how well you measure that protein (the smaller the quantity you can measure, the better)
|
|
|
ELISA uses what kind of well plates?
|
96 well plates
|
|
|
What are the types of ELISA?
|
Direct, Indirect, and Sandwich
|
|
|
What is the procedure before you use the ELISA?
|
immunize the animal with the antigen, then take a blood sample and prepare a serum to measure the antibodies you immunized for
|
|
|
What are some of the practical uses of ELISA?
|
you can compare antibody response. You can determine if a vaccine worked, and compare the levels of protection of different vaccines
|
|
|
What is titer?
|
is a measure of concentration. The titer is the highest dilution factor of an antiserum which results in a measurable response.
|
|
|
What is a substrate?
|
anything that creates color when bound to something
|
|
|
In ELISA, more color means...
|
more antibody bound to antigen
|
|
|
Indirect ELISA uses antibodies to bind to antigens or the reverse?
|
it uses antigens (added first) that bing to antibodies (added later)
|
|
|
What is the job of Bovine Serum Albumin in ELISA?
|
it prevents non-specific binding by blocking the remaining sites
|
|
|
What is the purpose of adding the serum sample in Indirect ELISA?
|
the serum sample contains the antibodies that binds to the bound antigen DUUUHHHHH
|
|
|
What is the main purpose of the Sandwich ELISA?
|
to measure antigen levels in a solution
|
|
|
What is an antibody?
|
It is a protein that binds to an antigen. eg. anticholera toxin antibody
|
|
|
Antibodies look like...
|
a Y and have identical binding sites and disulfide bonds
|
|
|
What is immunoglobin (Ig)?
|
this refers to the antibodies as proteins, regardless of what type of antigen it binds to.
|
|
|
What are the five types of antibodies?
|
IgG, IgM, IgA, IgD, IgE
|
|
|
What is IgG?
|
is the anibody found in highest concentration in serum
|
|
|
What is IgM?
|
This is the first antibody producer and it is made of five monomers, therefore it has ten binding sites (two per antibody, remember Y-shaped).
|
|
|
What is IgA?
|
this antibody is found in external excretions (tears, sweat, etc)
|
|
|
What is IgD?
|
this antibody is only found on the surface of B cells
|
|
|
What is IgE?
|
This antibody is found only on IgE B cells, basophils and mast cells
|
|
|
What are the functions of B cells?
|
proliferation after activation and production of antibodies after activation
|
|
|
What happens during B cell activation?
|
An antigen binds to the B cell surface Ig, then the B cell activates and begins to proliferate. Thereafter the B cell differentiates into a plasma cell. Finally, the plasma cell produces antibodies
|
|
|
What are polyclonal activators?
|
They activate subsets of B and/or T cells and they are not as specific. (LPS)
|
|
|
What are the three types of polyclonal activators discussed in class and which cell type do they activate?
|
Lipopolysaccharide activate B cells. Concavalin A & Phytohemagglutinin activate T cells. And Pokeweed mitogen activates both B and T lymphocytes.
|
|
|
What are lipopolysaccharide and where are they found?
|
LPS is found in the outer membrane of gram negative bacteria. E coli, Salmonella, Shingella. LPS are made of lipids and sugar bound together. LPS only activates IgM B cells and it results in IgM secretions and has very little effect on IgG.
|
|
|
Why do we need to activate B cells?
|
because we need to study activation of B cells to produce antibodies.
|
|
|
What is Coomassie blue?
|
it is the protein stain we use in SDS-PAGE.
|
|
|
On SDS-PAGE: How do we stop the antibodies from sticking to the nitrocellulose membrane?
|
We incubate the blot with Bovine Serum Albumin so that no other proteins can stick to the blot. This happens because the BSA saturates the nitrocellulose and there is no more space for other proteins to bind to it
|
|
|
Why do we stain the blot with the primary antibody? And what can we use to stain it?
|
Because in the blot everything is visible, we need to label the antibody. We can use a fluorescent compound, a radioactive compound or an enzyme.
|
|
|
Why do we use a secondary antibody?
|
Because we label the secondary antibody and this secondary antibody binds specifically to the primary antibody. The secondary antibody is use as a way to label the primary.
|
|
|
What is another name for the secondary antibody?
|
developing antibody
|
|
|
Why is the secondary antibody from a different species?
|
Because an antibody from a different species is guaranteed to create an immune response that would allow the primary antibody to bind to it(the secondary antibody)
|
|
|
Why do we prefer to use an enzyme, instead of a radioactive or fluorescent compound, to label the secondary antibody?
|
because we use the enzyme as an amplification system. If the enzyme substrate is uncolored but the reaction product is colored, then an enzyme could make many colored products which could be used to localize your specific protein. LONG ASS ANSWER, read the third paragraph on pg 58 of lab manual
|
|
|
Which are the most common enzymes that are coupled to secondary antibodies?
|
alkaline phosphatase (AP) and horseradish peroxidase (HRP) because these two enzymes can be isolated in large quantities.
|
|
|
what are BCIP and NBT?
|
these are the two substrates we used to act with alkaline phosphatase (enzyme coupled to secondary antibody).
|
|
|
How does alkaline phosphatase react with BCIP and NBT?
|
It hydrolyzes BCIP to remove a phosphate and this is then reduced by the NBT. The reduced NBT also precipitates and together they form a blue-grey precipitating stain.
|
|
|
Again... How do we stop the primary and secondary antibodies from binding to the blotted nitrocellulose membrane?
|
We use Bovine serum albumin
|
|
|
What does BCIP stand for?
|
5-bromo-4-chloro-3-idolylphosphatase
|
|
|
What does NBT stand for?
|
nitroblue tetrazolium
|
|
|
What is the color of the precipitate formed by BCIP?
|
Indigo and this color stays on the protein
|
|
|
What is the color of the NBT precipitate?
|
blue-gray
|
|
|
Which is the only band that stains?
|
the C band
|
|
|
EGF binds to... and this creates a...
|
The EGF-R. This activates the EGF receptor, which is a kinase. The EGF-R phosphorylates itself and other proteins with tyrosine residues. This activates a kinase phosphorylation cascade signaling pathway.
|
|
|
The control had phosphorylation because...
|
the control also contained proteins.
|
|
|
What is a synergistic effect?
|
Almost doubles the effect put together
|
|
|
What is the FACSCalibur?
|
is a general workhorse for a lot of clinical labs. Flow cytometer!!!!
|
|
|
Which are the heaviest and lightest molecular weights in the proteins we measured on the blots?
|
myosin is the heaviest (207,000Da) and the lightest is aporinin with 7,100Da. BSA was in the mids with 81,000Da.
|
|
|
What are the specs of the flow cytometer?
|
2 excitation lasers, 1000 events per second (one cell = one event), it can sort cells at 300 cells/sec
|
|
|
What are the types of lasers used by the flow cytometer?
|
argon laser at 488nm, and the red diode laser at 635nm
|
|
|
What is one of the problems with having the flow cytometer read a high number of cells at any given time.
|
The higher the number of cells, the higher the chance of counting two cells as one
|
|
|
What is the forward scatter?
|
this is how the size of the cell is determined. The forward scatter is the amount of light that passes through as a straight line
|
|
|
What is the side scatter?
|
this is used to determine the cell's granularity. This is the laser light bounces off to the sides
|
|
|
What are the stages of the cell cycle?
|
G0, G1, S, G2, M
|
|
|
What is happening when a cell is in G0?
|
Nothinng, the cell is in a quiescent state, not dividing
|
|
|
What is happening when a cell is in G1?
|
the cell is recovering from division and getting ready to do it again
|
|
|
What is happening when a cell is in S ?
|
Manufactoring of DNA
|
|
|
What is happening when a cell is in G2?
|
Finishing up DNA and then doubling it
|
|
|
What is happening when a cell is in M?
|
mitosis
|
|
|
What would you expect cells to be in the different cell cycle stages? 2C or C?
|
G0/G1 = 2C because everything is double up.
S varies between 2C and C. G2/M = 4C |
|
|
What are some of the DNA stains?
|
Hoechst, Dapi, Cyto Dye 24, and propidium iodide
|
|
|
What are the excitation and emmision wavelengths of propidium iodide?
|
excitation = 538nm but it can still be excited by argon at 488nm. Its emission is at 623nm.
|
|
|
What are some of the problems with using propidium iodide?
|
It is not specific for DNA because it also stains double stranded RNA, so we have to use RNase to rid of double stranded RNA. Also, it does not penetrate the cell membrane, so we have put little holes in the membrane using 70% ethanol to make the membrane solubilizable
|
|
|
Non-dividing cells are in the ___ phase.
|
G0
|
|
|
Cells beginning the preparation for cell cycle by synthesizing RNA and proteins needed for DNA replication are in the _____ phase.
|
G1
|
|
|
Cells in the S phase are...
|
actively synthesizing new DNA, and have twice as much DNA, but have not begun the actual process of separating replicated chromatids
|
|
|
Cells that begin the actual separation of the chromatids are in the process of ...
|
Mitosis (M)
|
|
|
How does the flow cytometer work?
|
it uses lasers to excite the fluorochromes on individual cells stained with fluorescent dyes and these are detected electronically by phootomultiplier tubes. It counts the number of cells and the amount of DNA in each cell
|
|
|
What does it mean for a cell to be euploid? aneuploid?
|
euploid = 2C
Aneuploid = not 2C. The chromosome number of an aneupliod is not an exact multiple of the haploid number. |
|
|
What is the name of the program used to collect the data from the flow cytometer?
|
CellQuest Pro
|
|
|
What is the name of the analysis program used to analyze the data from the flow cytometer?
|
ModFit LT
|
|
|
What is the option of gating in ModFit LT
|
This is used when the number of aggregates may be significant and may interfere with the analysis of the data. Gating allows to draw an area around just the data points you want to analyze.
|
|
|
What is % CV?
|
It is a quality of control term and It means the percent coefficient of variation. This is the accuracy of the S phase calculations. With normal 2C cells the %CV should be at most 8%. In tumor cells this percentage is higher
|
|
|
What is CELL#. When talking about the Quality of Control Analysis.
|
This is the number of events analyzed. Experts agree that these number should be at least 10,000 events.
|
|
|
What is the %BAD?
|
This is the percentage fraction aggregates and debris. This is the ratio of what the model has estimated as aggregates and debris to total the cell events in the histogram and should be less than 20%. Too many aggregates and debris would yield an unsaisfactory S-phase analysis
|
|
|
What is RCS?
|
Reduced Chi Square is a measure of how well the model fitted the data. Under 3 is a good fit, 3 to 5 is a fair fit, and over 5 is considered poor
|
|
|
What are some of the cytokynes produced by T cells that affect B cells performance?
|
T cells produce iL-4 and iL-6. iL-4 enhances proliferation in B cells and iL-6 enhances antibody production.
|
|
|
Name something that disrupts B cells.
|
Interferon inhibits B cell proliferation and antibody production
|
|
|
What does ELISA stand for again?
|
Enzyme Linked Immunosorbent Assay
|
|
|
What were one of the changes observed in the stimulated spleen cells on day seven?
|
they were more clumpled than the unstimulated because the were in the cell cycle, while the unstimulated were in G0
|
|
|
how can we infer about the health of a patient by simply looking at the leukocytes?
|
The number of leukocytes is fairly constant in the body of a healthy patient. These number fluctuate greatly in the presence of disease and infections.
|
|
|
Extensive bacterial infection results in elevated...
|
Neutrophils
|
|
|
Infection of the Epstein Barr Virus results in elevated ...
|
mononuclear cells (monocytes), therefore the name mononucleosis
|
|
|
Infections by parasitic worms result in elevated...
|
eosinophils
|
|
|
AIDS results in the depletion of...
|
T lymphocytes
|
|
|
How do we do a differential white blood cell count?
|
By making a smear of blood on s microscope slide, allowing it to dry and then staining the cells to count them.
|
|
|
Describe erythrocytes:
|
red blood cell, pink and depressed centers. No nucleus, and it carries oxygen to tissues
|
|
|
Define Plaletes:
|
Small cell fragments from megakaryocytes, involved in blood clotting and wound repair
|
|
|
Define Neutrophils:
|
polymorphic nucleus, faint blue cytoplams with granules. Major function is phagocytosis, and comprise 50-70% of total leukocytes
|
|
|
Define Eosinophils:
|
Polymorphic nucleus, intensely red/orange staining granules in the cytoplams. Fight parasites, about 1-5% of leukocytes
|
|
|
Define Basophils:
|
polymorphic nucleus. Black to blue staning granules in the cytoplasm. involved in allergic responses, release histamines. .5-1% of leukocytes
|
|
|
Define Monocytes:
|
Bean-shaped cytoplasm, pale blue cytoplasm which is often a "foamy" appearance, functions in phagocytosis and antigen presentation. 2-6% of leukocytes
|
|
|
Define Lymphocytes:
|
Round nuclei, usually with only a thin rim of cytoplasm. Include B (bone marrow) and T (thymus) cells and large granular lymphocytes. Functions in immune response, 20-30% of leukocytes
|
|
|
Name the types of cells found in the blood.
|
In order of numbers in the blood: Erythrocytes, neutrophils, platelets, lymphocytes, monocytes, eosinophils, basophils
|
|
|
Which cell type has a something to do with allergic responses?
|
Basophils
|
|
|
Which cell type fights parasites?
|
eosinophils
|
|
|
faint blue cytoplasm and has a major function in phagocytosis
|
neutrophils
|
|
|
Bean-shaped cytoplasm...
|
monocytes
|
|
|
Round nuclei and make up 20-30% of leukocytes
|
lymphocytes
|
|
|
involved in blood clotting and wound repair
|
platelets
|
|
|
What is the name of the stain we used to stain the blood smear?
|
LEUKOSTAT modified Wright's Stain (fisher diagnostigs)
|
|
|
What were the steps taken in the staning of the blood smear?
|
Dip the slide 5x into the blue colored fixative solution. Dip the slide 5x into the pink-colored Eosin stain. Dip the slide 5x into the purple-colored Methylene Blue/Azure A
|
|
|
What does CD stand for? And what is it used for?
|
Cluster differentiation number is the number given to the differnt proteins found on the surface of the different cell types
|
|
|
CD29
|
beta1 integrin subunit
|
|
|
CD49f
|
sigma6 integrin subunit
|
|
|
CD71
|
transfering receptor
|
|
|
CD220
|
Insulin receptor
|
|
|
CD4
|
Found on Helper T cells
|
|
|
CD8
|
Found on cytotoxic T cells
|
|
|
organ to right over the mouse spleen. And what is over this organ? (disection)
|
stomach and over the stomach is the liver
|
|
|
What did we use to activate the B lymphocytes
|
LPS - lipopolysaccharide
|
|
|
Concanavalin A (Con A) is a ____ and it activates _____
|
polyclonal activator and it activates T lymphocytes
|
|
|
Pokeweed mitogen is a ____ and it activates _____
|
polyclonal activatior and it activates B and T lymphocytes
|
|
|
Phytohemagglutinin is a ____ and it activates _____
|
polyclonal activator and it activates T lymphocytes
|
|
|
Dextran Sulfate (DxS) is a ____ and it activates _____
|
polyclonal activator and it activates B lymphocytes
|
|
|
What are the two types of T lymphocytes?
|
cytotoxic T cells and helper T cells
|
|
|
antigens are...
|
foreign subtances
|
|
|
What does MALT stand for?
|
mucosal associated lymphoid tissue
|
|
|
GIVEN: When analyzing the data from the flow cytometer: doblet peaks are wider peaks
|
given
|
|
|
GIVEN: the more cells the higher the peaks
|
g
|
|
|
GIVEN: the more dense the more DNA, the lower wider peaks
|
g
|
|
|
euploidy
|
true 2C
|
|
|
aneuploidy
|
not 2C (most cancer cells)
|
|
|
GIVEN 2C and 4C cells have the same size but will read different because...
|
the amount of chromatin
|
|
|
whats the beam splitter?
|
it separates the laser lights (green and red)
|
|
|
flow cytometer tells us about...
|
particle size, granularity, internal complexity and fluorescence intensity
|
|
|
fluidics
|
transport particles in a fluidic (saline solution) stram to the laser beam for interrogation
|
|
|
optics
|
the collection of components that steer the scattered and fluorescent light to appropiate detectors
|
|
|
electronics
|
convert detected light into proportional electronic signals
|
|
|
hydrodynamic focusing
|
the process whereby both the sample and sheath fluids move through a narrowing channel. The sample core remains in the center of the sheath fluid
|
|
|
what is the purpose of the excitation optics
|
to provide an energy source to excite fluorescent molecules
|
|
|
what is threshold?
|
is the minimim pulse height above which the signal will be processed
|
|
|
Cytotoxic cells...
|
kill virus infected or tumor cells
|
|
|
Helper T cells...
|
help b cells produce more antibody and help cytotoxic cells become killer cells
|
