Cell Bio Lab Exam 2

Methods to identify proteins

1. SDS PAGE - Molecular weight 2. Western Blot - Specific protein 3. Immunofluorescent staining -Specific protein

What do we label an antibody with?

A conjugated fluorescent compound or an enzyme. -FITC is a fluorescent tag that is green-yellow

Why do we block a Western Blot?

Blocking is done so that the antibodies do not stick non-specifically to the nitrocellulose because they are still proteins.

What are common CD designations?

CD220 = insulin receptor CD71 = transferrin receptor CD4 = helper T cells CD8 = cytotoxic T cells

How many chromosomes do various cell lines have?

Caco-2 = 96 chromosomes HT-289 = 68-72 chromosomes (hypotriploid) A549 = 64-67 chromosomes (hypotriploid)

What are exceptions to being diploid?

Cells undergoing meiosis Cells in M, S, or G2 phase of the cell cycle (between 2C and 4C). Abnormal cells

What is aneuploidy?

Cells with altered levels of DNA. Cancer cells have abnormal numbers of chromosomes or alterations in chromosomes

What are the experimental and control conditions in the immunoassay?

Experimental = cells stained with the FITC conjugated specific antibody Control = no antibody / FITC conjugated irrelevant antibody

What can we use to label an antibody?

Fluorescent compound Radioactive compound Bind an enzyme to the antibody Use a labeled secondary antibody

What is lipopolysaccharide?

Found on the outer membrane of gram-negative bacteria, like E. coli, salmonella, and shigella. Made of lipid and sugars bound together. - Our immune cells have keyed into this feature and we can recognize any. Nonspecifically activates B cells bc B cells have Toll-like Receptors (TLRs) to bind Only activates IgM B cells, resulting in IgM secretion. Very little effect on IgG

What are the different stages of the cell cycle?

G0 = cells not cycling G1 = can be cells recovering from division or preparing to initiate a cycle S = synthesis. Cells actively producing new DNA. There is a changing amount (increasing) of DNA as it goes through S phase. G2 = cells finished with DNA synthesis and have double the amount of DNA. M = cells actively in mitosis.

What are the pros and cons of faster and slower flow in the flow cytometer?

In faster flow, more cells can go through, but we may get two or more at a time. In slower flow, it takes more time, but we can be more assured that there is only one event at a time.

What is a laser scanning confocal microscope?

Instead of a light bulb, it uses lasers. -Narrow beam of light to excite the sample. - Fluorescent microscopy. A fluorescent light passes through a pinhole, which blocks all light except out of the plane of focus to avoid blurring from multiple focal plane.

What does the data from the flow cytometer look like?

It gives the number of cells counted vs relative fluorescence of the stained cells. Analysis using the LT program helps us decide if cells are in G0/G1, S, or G2/M phases. The plot of fluorescence intensity vs. number of cells has two peaks: G0/G1 phase is the first peak, G2/M phase is the second peak. S phase is between. The higher peak is the peak with the greater area.

What is the excitation of propidium iodide?

It has excitation wavelengths at 538 nm and 623 nm. We can excite the 538 nm fairly well by the 488 nm laser, so we won't be seeing the 623 nm.

What are neutrophils?

It is a polymorphonuclear leukocyte. Lobed nucleus. Cytoplasm has granules. Major function in phagocytosis. 50-70% of human leukocytes.

What are the problems with propidium iodide?

It is not specific for DNA and also binds RNA, so we need to remove RNA by RNase treatment. Cannot penetrate the cell membrane, so we must solubilize the cell membrane by adding 70% ethanol. Cannot be used with live cells.

Which polyclonal activators activate which cells?

Lipopolysaccharide activates B cells Concanavalin A and phytohemagglutinin activate T cells Pokeweed mitogen activates B and T cells

What do we expect to see in the Western Blot?

More bands, some heavier than others (more phosphorylated protein), and some that are in the EGF treated but not the control.

What is nice about DNA stain?

Most DNA stains fluoresce only when bound to DNA. Unbound stains have little if any fluorescence by comparison. So, there is no need to wash out the stain.

How do we identify cell types?

Most have something unique to that cell type. - B cells = anti-Ig antibodies - T cells = anti-TCR antibodies - Helper T cells = anti-CD4 - Cytotoxic T cells = anti-CD8

What can aneuploid cancer cells cause in cytometry?

Multiple peaks. This is also a helpful diagnosis for cancer.

Which surface proteins do not have a CD number?

Polymorphic surface proteins

What are antibodies?

Proteins made by vertebrates that bind to foreign proteins or antigens with high affinity and specificity. They are Y shapes with two binding sites and held together by disulfide bonds.

What happens in the flow cytometer?

The cell blocks the laser and scatters forward light. This is counted as an event (or cell). Laser hits cell and excites fluorochrome, and fluorochrome emits light.

What is the function of the lymph nodes?

The lymph nodes function to filter the fluid called lymph.

How do we know if cells are non-cycling/resting?

There is essentially one peak. All cells in G0 are resting. Best example comes from blood/spleen leukocytes.

What do we use the flow cytometer for?

To analyze cell cycle.

Where do we get B-cells?

We can get them from the blood. Wherever there are lymphocytes, there are B cells. They are also found in the lymph nodes. Also we can find them in the spleen.

What is the ploidy of most cells?

almost all normal cells are diploid with two copies of each chromosome (2C amount of DNA). Humans = 46 chromosomes

What are leukocytes?

white blood cells

What is euploid?

'Good ploidy' having an exact multiple of the number of chromosome humans = 2C = 46 chromosomes

How do we activate B cells?

1. Antigen binding to B cell surface Ig. -This only activates a few cells with an antibody specific for that antigen, so the response is low. 2. Adding antibodies to the B cell Igs - Expensive - Adding anti-mouse IgG to mouse B cells - Two binding sites on added Ab bind B cell BCR to each other - Activates more or all B cells 3. Polyclonal Activators - We use this - Agents that can activate whole groups of cells, regardless of what antigen they are specific for

What are the FACSCalibur excitation lasers?

1. Argon-Ion Laser -488 nm laser line. 2. Red diode laser -635 nm laser line.

What are the steps of a sandwich ELISA?

1. Coat with the capture antibody, add the specific antibody, and proteins stick to the plate 2. Wash 3. Blocking -block remaining sites with the bovine serum albumin to prevent non-specific bidding 4. Wash 5. Add antigen to the sample to bind to the capture antibody 6. Wash 7. Add enzyme-linked secondary antibody to bind to the antigen 8. Wash 9. Add substrate to produce color 10. Measure color produced by using microplane reader -More color = more bound secondary antibody-enzyme = more antigen in the sample

What are the uses of an indirect ELISA?

1. Compare antibody response -Determine if a vaccine worked -Compare levels of protection 2. Titer = reciprocal of the highest dilution of an antiserum which results in a measurable response

What are the different types of ELISAs?

1. Direct ELISA -Of little use 2. Indirect ELISA -Measure antibody levels in a serum -Immunize animals with antigen, take blood and make serum, measure antibodies (can be done for virus immunity). 3. Sandwich ELISA -Measures antigen levels in a solution (hormones, cytokines, antibodies, proteins) -Antibodies bind to an antigen -Enzyme linked secondary antibody detects the bound antibody

What are the different types of DNA stains?

1. Hoechst 2. DAPI 3. Syto dye 24 (not very good) 4. Ethidium bromide 5. Propidium iodide (we use)

How do we quantify proteins?

1. Scan SDS-PAGE or Western Blot bands to compare band density. 2. Specific assays for particular proteins or their ?

What is needed for a good assay?

1. Specificity -Measure only the specific protein in complex mixture 2. Sensitivity -Able to measure extremely small quantities of specific proteins (micro, nano, even pico) 3.Simple 4. Able to do many samples

What are the conditions of the background and reagent blank in the sandwich ELISA?

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Why do we do a reagent blank in the sandwich ELISA?

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What are B cells?

A lymphocyte type that produces antibodies. Has Ig as integral membrane protein - B cell helper -Binds antigen to activate B cell -Igs don't have CD number

What does fixing the cell do?

Adding ethanol solubilizes the membrane so propidium iodide can reach DNA and RNase can enter the cell.

What are problems with flow cytometry? How can we fix them?

Aggregates of two or more cells may occur. This can look like a large cell with 4C DNA. - We can use a slow flow rate for better distinction. - Also, doublet peaks are wider, so we can plot area vs. width to help us get rid of this (shorter width means it's probably a single cell going through, but if the width is higher, two singles going through).

What is the B-cell activation scheme?

An antigen binds to a BCR (B-cell surface Ig). The B cell becomes activate. B cell proliferates. -It'll go from G0 to G1, S, G2, M. Proliferation expands/amplifies the immune response. The B cell differentiates into a completely different cell type called a plasma cell, which is filled with RER to produce surface and secreted proteins (antibodies). Antibody secretion his exponentially higher after the second immunization or encounter with the antigen.

What is polyploidy?

An increase in the number of chromosomes

Antibody vs. Immunoglobulin

Antibody refers to the protein that binds to an antigen. -Anti-cholera toxin antibody. Immunoglobulin refers to the antibody as a protein, regardless of what antigen it binds to. It's more of a broad term. Immunoglobulin is the generic molecule; an antibody is used to talk about a specific one.

What are the lymphocyte types?

B cells - Produce antibodies T cells - Have TCRs that bind to antigen, but TCRs don't have CD number - Cytotoxic T cell = specific killer cells that kill virus infected or tumor cells. - Helper T cells = help B cells produce antibodies and help cytotoxic T cells become killer cells - Natural Killer Cells

What makes antibodies?

B lymphocytes

What flow cytometer do we use?

BD Biosciences FACSCalibur. It has two excitation lasers and can detect four emission colors. It can process up to 1000 events per second and sort 300 cells per second.

What are monocytes?

Bean shaped nucleus. Foamy cytoplasm. Function in phagocytosis and immune responses. 2-6% of leukocytes.

What are lymphoid cells?

Differential white blood cells. Aka lymphocytes. They have a round nucleus, thin rim at cytoplasm, and are resting cells. They function in the immune response. They make up 20-30% of total blood leukocytes. Always in G0 phase.

Which bacteria are gram negative?

E. coli, salmonella, and shigella. They have an inner membrane and an outer membrane. They have lipopolysaccharides.

What is an enzyme-linked immunosorbent assay?

ELISA uses a specific antibody to recognize and quantitate a specific protein, even in a complex mixture. It can be used for anything you can make an antibody for. It uses an enzyme-conjugated antibody for amplification and high sensitivity. It is simple and straightforward, and we can do 96 wells at once for many samples.

How do we study B cells?

Identify the B cell by Ig on the cell surface by immunofluorescence. So, we can look and see how many cells have IgG, IgM, IgA, etc.

What are the five types of immunoglobulins?

IgG: highest concentration in serum, so it is the one that really protects the body. It has the classic antibody structure with two heavy and two light chains. It is essentially a monomer and can be secreted or a B-cell receptor. IgM: made of five monomers held together by a J chain protein. It is the first antibody produced. Can be secreted or a BCR. IgA: found in external secretions, such as tears, saliva, nasal secretions, gastrointestinal secretions, urinary tract secretions, etc. It protects the moist, mucosal surfaces of the body. It can be secreted o a BCR. IgD: weird. Only found on B-cell surface as BCR. It is responsible for allergic responses. It can tell basophils and mast cells to produce histamine. IgE: found only on IgE B-cells, basophils, and mast cells.

Why do we do a differential WBC count?

Infection or disease can alter the counts. - High number of neutrophils = infection - High eosinophil = parasite - Sustained high levels = leukemia

What is the problem with SDS PAGE and Western Blot?

It does not show where the protein is

Why do we do a background in the sandwich ELISA?

It ensures that the secondary antibody does not bind to anything except the sample. It is usually subtracted from the samples and standard values.

How many CD designations are there?

Over 360

What are basophils?

Polymorphic nucleus. Blue and purple staining granules. Has a role in allergic responses. 0.5-1% of human leukocytes.

What are eosinophils?

Polymorphic nucleus. Red/orange stained granules. Fights parasites and some phagocytosis. 1-5% of human leukocytes.

What does the spleen cell culture look like in lab 11?

Prepare spleen cells. Add cells to 96 well culture plates. -Control: cell + media only -Experimental: cells, media, LPS -Compare activated by LPS and those not to see what produces the most antibodies Culture for 7 days to allow antibodies to collect in the wells. Harvest culture supernatant that contain the secrets Igs. Determine total IgM concentration using ELISA.

Why do we stain for cell surface proteins?

So we can identify cell types. -This works well for lymphocyte types and stem cells. Also, to see if and when cells express a surface protein. -Receptors, adhesion proteins. -Changes with the activation of the cell.

How do T cells govern B cell activation and antibody activation?

T cells produce cytokines that bind to or act on other cell types. They will tell the B cell to produce certain antibody types, or they may say to produce a lot more antibodies, or they may tell the B cell to differentiate into plasma cells. T cells and B cells interact through cell surface proteins to signal each other. This is the 2nd way how T cells activate B cells.

What is the function of the spleen?

The spleen filters the blood.

How does alkaline phosphatase work?

The substrate is BCIP. AP hydrolyzes BCIP to remove a phosphate. BCIP is then reduced by NTP to form indigo BCIP dimer that precipitates. This yields a blue-grey stain.

What is the goal of the western blot experiment?

To show that EGF has an effect on signaling within the cell.

Why do we need to activate B cells?

To study how B cells are activated to produce antibodies. To study how T cells govern B cell activation and antibody activation. To study how added agents can alter B cell activation and antibody production. -Maybe we want to increase a person's ability to produce a certain antibody. -How do T cell factors enhance B cell proliferation? -How do T cell factors enhance antibody production? -Effects of drugs

How do we identify proteins in a cell?

Use a specific antibody.

How do we estimate what part of the G0/G1 peak and G2/M peaks are really cells in S phase?

Use mathematical algorithms to estimate. Modfit LT will do this for us.

How do we determine where the protein is?

We can determine if the protein is inside the cell by immunofluorescence. -Solubilize the membrane with acetone and methanol to allow the antibody into the cell. We can determine if the protein is on the outside of the membrane by treating with antibody without solubilizing the membrane so the antibody never enters.

How do we see the antibody in a Western Blot?

We can directly label the antibody.

How can we look at the functions of B cells?

We can look at proliferation after activation using proliferation assays. We can look at the production of antibodies after activation.

How do we avoid problems in flow cytometry?

We can slow down the flow rate of the cells. We can plot FL-width vs. FL-area so we can gate around the G1/S/G2 cells, excluding doublets, triplets, etc.

How do we stain for proteins in the EGF pathway?

We can stain for tyrosine phosphorylated proteins. EGF stimulation should result in the tyrosine phosphorylation of several proteins. This shows that the pathway has been activated, which is our goal.

What are the steps for staining before analysis in flow cytometer?

We fix the cell, centrifuge it, re-suspend it in buffer containing propidium iodide and RNase, incubate for 30 minutes, then analyze.

How do we know if cells are in cycle?

We get a second peak at 4C for G2/M phase at about double the fluorescence intensity of the G0/G1 phase.

How do we block in a Western Blot?

We incubate the blot with bovine serum albumin so no other proteins can stick to the blot non-specifically.

How can we address doublet discrimination?

We plot fluorescence-width vs. fluorescence-area instead of just fluorescence vs. cell count. Doublets are easily seen apart from single G2/M cells. Draw a gate around the G1/S/G2 cells and only analyze the cells inside the gate to exclude doublets, triplets, etc. It also excludes fragments.

How do we do a differential white blood cell count?

We prepare a smear of blood on a slide, stain the smear with the leukostat stain, count 100 leukocytes, and take percentage of each leukocytes type.

How can we tell what cycle a cell is in?

We stain the DNA and get three populations of cells. 1. G0/G1 with 2C amount of DNA. 2. S with varying amount of DNA. 3. G2/M with 4C amount of DNA

In Western Blot, why do we use antibodies and not a stain?

We use antibodies because a protein stain would stain all the proteins on the blot without telling us which is ours.

What enzyme and substrate do we use in the sandwich ELISA?

We use peroxidase conjugate goat anti-mouse IgM antibody, but we could have also used alkaline phosphatase. The substrate is ABTS. It is colorless, but with H2O2 creates ABTS-green and water.

Why do we use paraformaldehyde?

We use this in the immunoassay experiment to stain because it allows us to analyze cells any time up to 2 weeks later.

What enzyme did we use to link to the secondary enzyme.

We used alkaline phosphatase.

What did we use to label the antibody in the lab during the Western Blot?

We used an enzyme-conjugated goat anti-rabbit immunoglobulin antibody. This binds to the first antibody. An enzyme is linked to it.

What is ELISA?

enzyme-linked immunosorbent assay

What does forward and side scatter of light indicate?

forward scatter = indicates cell size. side scatter = indicates cell granularity.

What is the equation of the standard curve we get from the ELISA?

log (y) = a*log (x) + B a = slope B = y intercept

What are erythrocytes?

red blood cells. They are pink with depressed center, no nucleus, and carry oxygen to tissues.

How does the EGF pathway work?

EGF binds to the EGF receptor, activating the EGF-R, which is a kinase. EGF-R phosphorylates itself and other proteins at tyrosine residues. This sets off a kinase tyrosine phosphorylation cascade signaling pathway.