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81 Cards in this Set
- Front
- Back
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trypan blue
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used in cell counts for the determination of viability
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cells/mL =
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avg. number of cells per large square x 10^4 x 10
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viability determination
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% viable cells = (number of viable cells/total number of cells) x 100%
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how was KZH created?
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By using a T-cell myeloma fusion partner that was stably transfected with a reporter construct containing the LacZ gene under transcriptional control of a the IL-2 promoter. The myeloma was fused with antigen specific spleen cells from a HEL/BSA immunized mouse to creat the KZH T-cell hybridoma
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Protein assay
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used to determine how much protein is in an unknown sample
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Key features of the myeloma parent cell line used in class and why its used in making hybridomas
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1. permanently growing
2. HBPRT - (can be selected against using HAT medium) 3. does not secret antibody |
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HAT
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hypoxanthine aminopterin thmidine
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What does aminopterin do?
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Blocks the main biosynthetic pathway for purines/pyrimidines
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What does the conditioned media made in the cell fusion experiment consist of?
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6 mL of saved myeloma supernatant and 6 mL of IMDM-20-2XHAT medium
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ELISA
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enzyme linked immunosorbent assay
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Steps in an ELISA
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1. polystryene coated with antigen
2. sites on the plastic surface still capable of binding protein are blocked by addition of an unrelated protein such as Blotto 3. Removal of excess antibody and any other unbound serum proteins by washing 4. detection with addition of an enzyme conjugated second antibody which binds to the first antibody 5. amount of enzyme-conjugated antibody is measured by the ability of the enzyme to hydrolyze a colorless substrate to yield a colored product. |
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ELISA controls
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1. no antigen or no reagent controls - wells coated with PBS are no antigen (no BSA./HEL) controls. demonstrate reactivity is only found in presence of antigen
2. unrelated antigen for antigen specificity controls - wells coated with unrelated antigen should not have reactivity 3. normal mouse sera as negative control - nromal mouse sera from unimmunized mice should not bind to antigen-coated wells. 4. polyclonal mouse sera - demonstrates assay is working 5. soluble antigen inhbition controls to positively demonstrate antigen specificity - binding of antibody to antigen coated wells can be inhibited or blocked by prior incubation of the antibody witht eh same antigen as being tested in the ELISA plate |
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Antigen caputre ELISA
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Also known as sandwich ELISA; detects antigen instead of antibody
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Poisson distribution
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if an average of 1 cell is plated per well, 37% of the wells will contain 0 cells and show no growth while 63% of the wells will contain one or more hybridoma cells and will tehrefore show growth if cloning efficiency is 100%.
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what is the cloning medium used in limiting dilution clonging?
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IMDM-20 (20% FBS) with 10% conditioned medium
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immunoprecipitation
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used to isolate and characterize a particular cellular constituent from lysates of whole cells
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why must the lysate be precleared in immunoprecipitation?
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To remove any materials in the lysate that non-specifically bind to agarose beads of IgG.
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What does the lysis buffer used in the immunoprecipiation contain?
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20mM Tris-HCl, NaCl, Nonidet P-40, bovine serum albumin, EDTA, leupeptin, and aprotinin.
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What are leupeptin and aprotinin?
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Protease inhbitors
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what does the control cell lysate in the IP consist of?
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Unbiotinylated A-1 cells
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SDS
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sodium dodecyl sulfate; SDS binds to proteins and denatures them and covers them with negative charge
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DTT
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dithiothreitol; reducing agent that breaks disulfide bonds to -SH groups
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2-mercaptoethanol
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reducing agen breaks disulfide bonds to -SH groups
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what is the purpose of the glycerol/sucrose in SDS-PAGE?
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added to the sample buffer to provide increased density to the samples so that they will sink in water
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what is the purpose of the bromophenol blue?
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bromophenol blue is added so that the samples will be easily visible and to provide a dye front to follow as the gel runs.
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APS and TEMED purpose
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to polymerize gel (APS = ammonium persulfate)
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Western blot
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employs an immunochemical probe to demonstrate the presence of a particular antigen in a mixture of proteins.
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what is the substrate used in the Western blot?
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NBT/BCIP
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what does streptavidin bind to?
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all biotin labeled proteins
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flow cytometry
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used to analyze and or seperate individual cells that have been stained with antibodies conjugated to fluorescent dyes
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forward scatter
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measures cell size
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side scatter
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measures cell granularity
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propidium iodide
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DNA stain that is excluded form living cells
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how do we inhibit capping and endocytosis in flow cytometry?
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By using sodium azide and added it to the staining buffer and by keeping the cells on ice
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hemacytometer
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specialized glass slide where cell counts are done
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thymus
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primary immune organ wehre T-cells mature
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spleen
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secondary immune organ that filters the blood
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optimal cell culture condition
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37C, 5% CO2, high humidity
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phenol red
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added as a pH indicator
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what does conditioned media contain?
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usually uncharacterized but probably contains many different growth factors including cytokines and hormones.
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why do we use ribi adjuvant?
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inorder to increase the innate inflammatory response and therefore tirgger a more vigorous adaptive immune response
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PEG
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polyethylene glycol - highly hydrated polymer added to enhance fusion by inducing agglutination and cell to cell contact
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what are some uses for monoclonal antibodies?
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1. purification of proteins
2. identification of cell populations 3. diagnostic 4. immunotherapy |
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what are the steps in T-cell activation?
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1. signal 1 - t-cell recognition or binding to antigenic peptide presented on MHC of APC
2. signal 2: co-stimulation: APC stimulates T-cell via co-stimulatory molecule |
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Direct T-cell proliferation assay
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This is a radioactivity assay that uses a tritiated nucleotide (3H-thymidine) which is incorporated into replicating DNA. Growing cultures of cells are then incubated with 3H-thymidine. Cells are lysed and DNA is harvested onto fiber filters. The amount of radioactivity is measured in counts per minute using a scintillation counter
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Procedure for MTT assay tests for cytokines in supernatants using a cytokine dependent cell line
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Assay uses cell lines called CTLLs which are dependent on IL-2 to grow. To make an IL-2 positive control curve, CTLLs are placed in culture with known amounts of IL-2. The resulting CTLL proliferation is compared wtih test supernatants from activated T-cells. MTT is added to the CTLLs and is cleaved by mitochondria to form a dark blue insoluble formazan crystal. The amount of lbue color product is detected by a microtiter plate reader and used to correlate with the amount of cell proliferation
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IL-2 ELISA
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measures amount of IL-2 protein in activated T-cell culture supernatants
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IL-2 intracellular staining done by flow cytometry
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Stain T-cells stimulated with antigen for production of IL-2; activated T-cells are premabilized with detergent and stained with fluorescently labeled antibodies against IL-2. Assay determines the cytokine production in specific cells in contrast to other assays which examine cultures as a whole
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Brefeldin A
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drug that disrupts golgi membrane and allows access of antibodies to cytokines released from vesicles into the cytoplasm.
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copper dependent protein assay
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include biuret, Lowry, and BCA assay. Color in BCA assay is the result of the formation of Cu+2 protein compplex under alkaline conditions followed by reduction of Cu+2 to Cu+. The amount of reduction to Cu+ is proportional to amount of protein present
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coomassie blue dye binding assay
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also used in protein assays. Coomassie blue dye binds to protein molecules in acid pH and shifts color spectrum visibility from 465 nm to 595 nm.
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what are some problems with protein assays?
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1. detection range and accuracy - most proteins assays have a detection range between 20 ug/mL and 1000 ug/mL
2. reproducibility - protein to protein variation causes reproducibility to be a problem 3. detergents - can interfere and cause a lot of background 4. preparations - often obscure color changes and can also throw off accuracy of a protein assay |
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what are some uses for ELISA?
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1. measuring protein after purification
2. quantifying secreted protein production 3. screeining hybridomas 4. determine past or present exposure to infectious agents 5. determine presence of infectious agents, hormones or cytokines in the blood |
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angtien coat ELISA
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measure amount of antigen-specific antibodies in a sample
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antigen capture ELISA
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measure the amount of an antigen
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polyclonal sera
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commonly used as the coating antibody reagent for max. antigen capture. For antibody detection in antigen coat ELISAs, secondary detection reagents are often polyclonal because they provide amplification signal since each primary antibody can have many secondary antibodies bound
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AP-GAM
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alkaline phosphatase conjugated goat polyclonal antisera against mouse IgG
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monoclonal antibodies
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used for detection antibodies because they can be selected for high affinity and specificity
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what are some factors affecting the success of single cell cloning?
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1. issues of technique and reagents - pipetting errors, dilution errors, and calculation errors
2. stability of cells - hybridomas are very fragile and sometimes will die for no apparent reason and may also lose their capacity to make antibody. 3. distribution of cells in wells - see poisson distribution 4. cloning efficiency |
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cloning efficiency
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how well a cell line grows from a single cell or what percentage of the singel cell wells will grow.
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what are some uses for immunoprecipitation?
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1. isolate a small amount of protein
2. demonstrate the presence of a protein on the surface 3. identify protein protein interactions |
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why do we need to lyse the cells in the immunoprecipitation/
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to release the antigen from the membrane even for surface proteins
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what are some methods to lyse cells?
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1. sonication
2. enzymes 3. glass beads 4. detergent lysis |
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what is the advantage/disadvantage of polyclonal antibodies in immunoprecipitation?
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Advantage: more epitopes on the antigen can be bound
Disadvantage: potential for non-specific binding to irrelevant proteins resulting in hier background. |
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what are some methods for purification of immune complexes?
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1. aggregate precipitation - antigen antibody complexes may cause large aggregates to form and may be pelleted by centrifugation at high speeds
2. beads - solid phase method to the immune complexes to separate the ag/ab complexes by centrifugation 3. protein A and protein G - bacterial cell wall components that non-specifically bind to Fc protion of antibodies. |
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co-immunoprecipitation
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extension of the IP technique used to determine whether a given protein physically interacts with other proteins. Can be used to ID the presence of a known protein by Western Blot with specific antibody or ID novel proteins taht interact with a known protein as determined by SDS-PAGE and protein staining
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what does the sample buffer in SDS-PAGE consists of?
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1. SDS
2. DTT or 2-me as reducing agents 3. bromophenol blue - visualization |
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what does running buffer do and consist of?
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running buffer allows for recirculation of the charge; consists of tris and glycine which helps in the transfer of the charge
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what is the primary goal of scientific writing?
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to further scientific knowledge that can only be achieved by describing and sharing results iwth others
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what are the 5 sections in research papers?
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1. introduction
2. materials/methods 3. results 4. discussion 5. abstract |
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introduction
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explains why you did the experiments
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materials and methods
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how you did the experiments
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results
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what did you find out from doing the experiments
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discussion
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why your data is important
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what are the four parts to a figure legend?
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1. title
2. experimental details 3. definitions 4. statistical information |
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cell surfae staining
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antibodies to cell surface markers are used to ID cell populations by measuring surface expression of receptors such as CD4, CD8, CD3, CD14, etc.
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intracellular staining
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premeabilization of the cells can be done to stain cytoplasmic antigens. These antigens are usually signaling moelcules such as NF-kB or proteins that will be secreted such as cytokines.
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how is permeabilization done in intracellular staining in flow cytometry?
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usually done with detergents such as Saponin or Tween.
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hydrodynamic focusing
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sample is injected into center of a sheath flow, the combined flow is reduced in diameter forcing the cell into the center fo the stream in attempt to get each individual cell into a signel droplet as it passes the laster. As each droplet containing your cells of interest intercepts the light source or laser, it will scatter light and fluorochromes that can be excited to a higher energy state
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photomultiplier tube
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sensor that converts light energy into electrical current and generates a voltage pulse signal
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color compensation
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subtraction of a percentage of the signal from one fluorescent light sensor detector from the signal of another fluorescence light sensor to correct overlap of one dye's excitation measurement
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