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SC BIOL 425 - chapter_3_powerpoint

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Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Human Cell CultureHuman Cell CultureHL-60 cellsHL-60 cells differentiateSlide 11Slide 12Slide 13Slide 14Slide 15Counting Cells to Determine Cell ConcentrationExample CalculationToday’s exerciseChapter 3Anatomy of a Light MicroscopeOcular LensesRotating HeadNosepieceObjective lensesArmBaseStageCondenser/DiaphragmFine AdjustmentCoarse AdjustmentIlluminatorDimmerPlease use the proper procedure to set up and put away the microscopes!Exercise: Blood Cell Identification using a light microscope•Practice bringing a slide into focus (bring up to 40x lens, 400x total magnification)•Human Blood Smear slide; see pictures in manual to identify one of each of the following:MonocytesGranulocytesErythrocytesHematopoiesisIn order of descending prevalence, you will see: erythrocytes, neutrophils, basophils, eosinophils , monocytes, and thrombocytes.Image source: http://en.wikipedia.org/wiki/File:Hematopoiesis_simple.svg•IDENTIFY 3 BLOOD CELL TYPES FROM HUMAN BLOODone monocyte, granulocyte, and erythrocyte(You should be able to identify these cells from memory before you leave today!)Remember:1. Always focus on the 10X objective and then go up to 40X objective.2. Do not use the 100X objective.3. DO NOT put prepared slides on top of the hemacytometer.Today’s exerciseMutipotentTotipotent: able to form every type of cell in the bodyPluripotent: able to form most cell types, but not limited to a specific systemMultipotent: able to form multiple cell types, but restricted to a specific lineage/system= Pro-myeloid cell, ie HL-60 cellWhite Blood CellsRed Blood CellsNerveBoneOther TissuesHierarchy of Stem Cells TotipotentPluripotentBlood Stem CellsOther Stem CellsMuscleHuman Cell CultureCancer cells are isolated from a human andgrown in culture•Normal cells (primary cells) grown in culture die after 50-100 divisions•These cells have undergone changes (transformations) which cause them to become IMMORTAL so they may be maintained indefinitelyInactivation of a tumor suppressorActivation of an oncogeneViral infection•These immortal cells are a cell lineHuman Cell Culture•Cells are maintained in a media that contains all of the nutrients the cells need to survive•They are maintained in an incubator that provides them with the proper temperature and carbon dioxide content in the environment•HL-60s are a suspension cell line, meaning they do not adhere to solid surfacesHL-60 cells•HL-60 cell lines was derived from the blood cells of a 36-year-old Caucasian female with acute promyelocytic leukemia•HL-60 cells are myeloblastic (promyelocytic)•They require simple maintenance in vitro and grow in suspensionHL-60 cells differentiate•Environmental conditions, like pH and chemical inducers, can facilitate differentiation of these cells along the myeloid path•They are commonly and easily differentiated in vitro with the use of chemicals into neutrophils or monocytes•As HL-60 cells differentiate they cease proliferation and begin to:•Express new genes and molecules•Undergo morphological changes•Enter apoptosisA hemacytometer is a special kind of slide that is generally used to measure the density of cells in a culture.View from sideThicker than a normal slideView from TopChambers on either side where the cell culture is loadedGrids allow you to count the # of cells in a specific area.Exercise 1: HemacytometerHemacytometer Grid1mm1mmEnlarged view1. Place coverslip flat on top of raised grid area2. Add dyed cell suspension (well mixed) using your micropipette, so that the suspension is pulled underneath the coverslip3. Place hemacytometer on the microscope stage. Bring grid into focus on lowest magnification setting first. Bring up to next magnification, so that you can see the cells. Count the live cells.Loading cell suspension on a hemacytometerwww.bme.gatech.eduTrypan Blue Staining: Live/Dead Assay:•The hemacytometer grid is divided into 9 large squares, each with a surface area of 1 mm2 and a depth of 0.1 mm when the coverslip is in place.1 mm2 X 0.1 mm X 10* (correction for depth) = 1 mm3•If you count the total number of cells in the four large corner squares plus the middle square, you will have the cell number in a 0.5 mm3 area.5 X 0.1 mm3 = 0.5 mm3 •There are 1000 cubic micrometers in one cubic millimeter (which is equal to a microliter).1000 mm3 = 1 mm3 = 1ml1000**ml = 1ml•You may then calculate the number of cells using the hemacytometer, use the following formula:(average number of cells/mm2)(10*)(1000**)(dilution factor) = # of cells per milliliter1mm1mmCounting Cells to Determine Cell Concentration•Mix 0.2 ml cell suspension with 0.2 ml Trypan Blue solution•Count the live cells in 4 or 5 squares, take the average •Multiply the average by 2, to account for the trypan dilution (0.5X concentrated 1X concentrated)•Multiply by 104 for the amount of cells in one mL of cell suspensionExample Calculation•What is the density of your cells in one milliliter of cell suspension? •If you counted your cells and your average number of live cells in 4 squares is 25: 25 x 2 = 50 (number of cells in a 1X concentrated solution)50 x 104 (number of cells in one mL of cell suspension)•Answer: 50 x 104 (5 x 105) cells/mLToday’s exercise•Determine the concentration of viable cells per mL in your cell sample by performing a Trypan Blue viability


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