2814406_1834208486_LabManual.pdf

2814406_1834208486_LabManual.pdf - 5 Count the viable...

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5. Count the viable cells (not blue) lying within the four 1 mm squares labeled b, c, d, and e (Figure 1B). Do not count the blue stained cells because those are non-viable cells. Note 1) It is recommended to make a rule such as counting cells on top and left-hand lines of each square, but not those touching the bottom or right-hand lines, in order to avoid double-counting. Note 2) Recommended cell density is 20~50 cells/square. Appropriate cell density can be obtained by adjusting dilution factor. To calculate the concentration of the cells, first calculate the average of four 1 mm 2 areas and then apply this formula: Cells per mL = the average count per square × dilution factor 10 -4 mL Ex. If the average cell count per square is 45 cells, (45 cells × 2)/10 -4 mL= 9 × 10 5 cells/mL 6. Clean the hemocytometer by rinsing with water and drying thoroughly with a soft wipe. A. B. Figure 2.4. Hemocytometer; A ) adding a cell suspension to an assembled slide, B) magnified view of the grid.
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LAB 4: Cytotoxicity of cancer drugs Apoptotic assay Objective: In this experiment, students will learn how to quantify cell viability based on apoptosis by conducting cytotoxicity tests of cancer drugs using an apoptotic assay. Students will also gain an introduction to the basic principles of flow cytometry. Students will learn: hallmark traits of apoptotic cells and how it can be used to quantify cell viability basic principles of flow cytometry and its different applications Introduction: Flow cytometry: Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a narrow, rapidly flowing stream of fluid. The flow cytometer allows the analysis of multiple parameters of single cells by passing thousands of cells individually through a laser beam within a small time frame and capturing the light that emerges from each cell as it passes through (Fig. 4.1). The vibrating mechanism causes the stream of cells to disperse into individual droplets. The system is specifically adjusted so that there is a low probability of more than one cell being in a single droplet. There are a number of detectors in place; one in line with the light beam (Forward Scatter or FSC) and several perpendicular detectors (Side Scatter or SSC). FSC correlates with the cell volume (size) and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). The data gathered can be analyzed statistically by flow cytometry software to report cellular characteristics such as size, complexity, phenotype, and viability 11 .
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What is apoptosis? Apoptosis is one of the main types of programmed cell death which involves a series of biochemical events that lead to many morphological changes 12 . These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, as well as alterations in the cell membrane, such as loss of membrane integrity and attachment.
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