endocytosis Lab questions need help on EXPERIMENTAL PROTOCOL: This exercise will
ID: 196393 • Letter: E
Question
endocytosis Lab questions need help on
EXPERIMENTAL PROTOCOL:
This exercise will engage students in examining uptake of green fluorescent-tagged bacteria by macrophage cells and unveil the endocytic mechanism used by them. Half of the class will assess bacterial endocytosis by macrophages at different time points, while the other half of the class will test the impact of a endocytosis-altering compound on bacterial uptake.
1.) This experiment compares a treatment condition against a control. What type of control
is used in this exercise, and why is it important to run control to begin with?
2.) What is a fixative, and why is that needed as a part of this exercise?
3.) Given what you know about the mechanism you observed today, what kind of chemical inhibitor do you think would work best to disrupt that process? What cellular feature Latruncullin A would ideally target?
4.) The mechanisms of phagocytosis and macropinocytosis share a number of molecular features. In this exercise, how do you know which process the macrophage is doing?
Explanation / Answer
Question 1.
Answer. Positve and negative control should be used in this experiment.
Positive control : Something that is definitely going to be internalized by macrophages.
Negative Control : Something that is not going to be internalized by macrophages.
Test : Bacteria Tagged with GFP + Macrophages.
It is important to run both positive and negative control, to check the fidelity of the experiment performed. Positive control tells that everything is fine and experiment has worked. Negative control tells that the result is specific and is not just a random outcome.
Question 2.
Answer. Fixatives are chemicals that helps in preserving biological samples from decay due to autolysis or putrefaction. e.g. Formaldehyde.
Importance of using fixatives :
Question 3.
Answer. Cytoskeleton movement is required for endocytosis, so chemical inhibitors targetting cytoskeletal proteins(Microtubules, microfilament or intermediate filament), particularly actin cytoskeleton, would be effective to disrupts this endocytic process. e.g. Cytochalasin.
Latrunculin A targets actin monomers near the nucleotide binding cleft with 1:1 stoichiometry and prevents their polymerization.
Question 4.
Answer.
Phagocytosis of pathogens by macrophages initiates the innate immune response, which in turn activates adaptive response. In order to discriminate between infectious agents and self, macrophages have evolved a restricted number of phagocytic receptors, like the mannose receptor, that recognize conserved motifs on pathogens. These receptors induce rearrangements in the actin cytoskeleton that lead to the internalization of the particle.
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