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How are cells put into culture?
Primary and Sub-culturing
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What is primary culture?
1. Explant culturing: small pieces of tissue are attached to a culture dish and bathed in culture medium. 2. Enzymatic dissociation: speeds up process by adding digesting enzymes to dissolve the cement holding cells together. They are then placed into a medium.
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What are the different types of cell culture?
Fibroblast-like, epithelial-like, and lymphoblast-like
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What are Epithelial-like cell cultures?
- cells that are attached to a substrate and appear
- flattened and polygonal in shape.
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what are Lymphoblast-like cell cultures?
Cell that don't attach normally to a substrate but remain in suspension with a spherical shape
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what are Fibroblast-like cell cultures?
- cells that are attached to a substrate and appear
- elongated and bipolar, frequently forming swirls in heavy cultures.
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What are the optimum conditions for cell growth?
Controlled temp, Good substrate for cell attachment, appropriate medium, incubator (maintains pH & osmolality)
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Describe how you would practice sterile technique.
Gloves, Pre-sterilze(alcohol), Prevent wafting, Sterile pipets, Fume hood
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Describe the steps of enzyme cytochemistry
Incubation with a special colorless enzyme substrate
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what is Fixation?
Fixed by immersion in ethanol or acetone. Makes them permeable to stains. Causes cells to adhere to slides and fix them in place
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What is Incubation with a special colorless enzyme substrate
- Enzyme catalyzes the conversion of the subrate to a
- colored product
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what is Detection of the colored product by microscopy?
Colored product deposited at the site of enzyme action.
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Describe how you visualized the location of specific enzymes. Are you directly visualizing the enzyme itself?
- Enzyme action is colored and allowed for visualization
- via microscope. Can not visualize the enzyme itself, just its location
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What type of receptors did you study in lab? How did you detect the location of these receptors in epithelial cheek cells?
- Cell surface receptors and Lectin receptors
- observed the purple staining indicated surface receptors. detected lectin receptors by clumping
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How did you determine what sugar residues the ligands are binding to on the receptor?
coloration and clumping
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