Immunology Basic

how we study cells hypothesis driven experiments cell culture the technique used to grow cells or tissues outside the organism under strictly controlled conditions How are cells isolated from tissues? breaking down the cell-cell and cell-matrix interactions (use mechanical fragmentation, trypsin or EDTA) cell growing environments supplied with proper nutrients (amino acids, minerals, vitamins, salts, glucose, etc.), serum (insulin and growth factors) and grown at 37 degrees in a CO2 incubator trypsin an enzyme that non-specifically cleaves proteins (acts as a monomer that cleans the outside of the cell) EDTA ion chelator that chelates iron or magnesium that decreases the functioning of sticky proteins purpose of incubators maintain temperature, humidity and [CO2] why is CO2 important in incubators CO2 interacts with carbon and salt in the media which is very important for maintaining pH (~7-7.4) primary cell culture Refers to cells taken directly from an organism. These cells usually divide a limited number of times (~50 generations) and also undergo contact inhibition cell line refers to cells which are transformed and are able to grow indefinitely (immortal cells) and are less likely to exhibit contact inhibition first human cell line HeLa - from cervical carcinoma biopsy of Henrietta Lacks Morphology of normal fibroblasts - elongated - aligned - orderly packed - grow in parallel arrays - contact inhibition Morphology of transformed fibroblasts - rounded - hair like processes - disorganized - grow one atop other - loss of contact inhibition embryonic stem cells (ES) pluripotent and can be grown indefinitely in culture under appropriate conditions stem cells can give rise to other cells that are like a stem cell but are more restricted in cells that it can subsequently give rise to (asymmetrical division is much more common) progenitor cells more restricted in what it can give rise to (only a couple) fibroblast cells produce growth factors and other types of proteins which help to grow these inner mass cells pluripotent cells give rise to the 3 types of germ layer cells adult stem cells - required to maintain and repair the tissue - capable of generating a limited number of different cell types - adult stem cells are located in a stem cell niche (adjacent cells which provide signals to either self renew or differentiate) - multipotent multipotent (not pluripotent) - can only give rise to a small number of different cell types - help to replenish worn out tissues niche cells help provide signals to the stem cells to tell them to divide more and generate more stem cells or to generate more differentiated epithelial cells (which can be used to replenish the lost cells that occur at the tip of the villi) asymmetric division of stem cells useful for maintaining stem cell reserve an generating differentiated cells (most common way to maintain the stem cell reserve throughout adulthood )--> in embryonic development, more differentiated cells are needed to form the fetus symmetric division of stem cells creates more adult stem cells stem cells divisions must meet 3 objectives - maintain the stem-cell population - increase the number of stem cells - produce cells that go on to differentiate reprogramming of fibroblasts can be accomplished by introducing into differentiated cells, the Yamanaka Factors: Yamanaka Factors three genes that are characteristic of ES cells (Oct4, Sox2, Klf4) and one frequently expressed in cancer cells (c-Myc) induced pluripotent stem cells (iPS cells) pluripotent stem cells obtained from differentiated normal cells asymmetric cell division two different types of daughter cells are formed from one parent cell symmetric cell division both daughter cells formed in symmetric cell divisions are identical, but may have different fates if they are exposed to different external signals resolution the ability to distinguish between two very closely positioned objects as separate entities formula for resolution D = (0.61 lambda)/ (Nsina) Nsina numerical aperture (higher is better) HAT medium allows regular spleen cells to survive but does not allow myeloma cells to survive, therefore after many generations, only the hybridoma cells will remain