lab report questions

Question

Question 7: summarise the results of your phagocytosis assay, comparing the differences, if any, between what you observe for cells with beads A and cells with beads B.

beads A x10 objective:

beads A x25 objective

beads B x10 objective

beads B x25 objective

Question 8 [short Text answer]

Marked out of 15.0

Using either your own cells with beads A or B, or the online images of the same, calculate a phagocytosis index for each treatment. Give the numbers of cells counted, numbers of beads etc and the final PI numbers as mentioned in the practical handout.

Question 9 [Text answer: Up to 250 words]

Marked out of 15.0

Beads A are clean beads , whereas beads B were pre-coated in surface layer of mouse immunoglobulin G (IgG). What differences might you expect (whether your experiment worked or not) upon exposing the mouse RAW 264.7 macrophages cells to these different beads. (do not give a mechanistic reason for it here, just what you might expect to happen)


Question 10 [Text answer: up to 250 words]

Marked out of 15.0

What immunological mechanism(s) might be involved in altering phagocytosis given your knowledge of the beads and cells, irrespective of whether you saw in your own experiment?

Answer 1

You’ll need to count the number of internalized beads and the number of macrophages you observed. Phagocytosis Index (PI) = Total number of internalized beads / Total number of phagocytic cells.

Expected Results: Beads coated with IgG are expected to have a higher PI compared to uncoated beads. This is because IgG acts as an opsonin, a molecule that tags particles for phagocytosis.

Notes:

IgG-coated beads → have a tag → easier to grab → more phagocytosis → higher PI

IgG-coated beads = are likely to be phagocytosed more readily. The beads mimic antibody-tagged pathogens, which macrophages detect and engulf. So, you will see a higher number of beads internalized per cell, and more cells participating in phagocytosis, compared to uncoated beads.

Uncoated beads → no tag → harder to recognize → less phagocytosis → lower PI

Clean (uncoated) beads = phagocytosis is expected to be relatively low. The cells may struggle to recognize or bind these beads efficiently.

Immunological Mechanisms Behind Phagocytosis Differences:

Simple summary: Opsonization means tagging something so the immune system can find and engulf it more easily. In this case, IgG antibodies stick to the surface of the beads. Macrophages have special receptors that can grab onto these IgG tags. Once the bead is tagged, the macrophage knows to engulf it.

More detailed mechanism: Beads are covered in IgG antibodies, which are recognized by Fc gamma receptors (FcγRs) on the surface of RAW 264.7 macrophages (a mouse immune cell line commonly used in research). Macrophages are more likely to engulf particles that are coated with IgG antibodies than ones that are clean or uncoated. Macrophages have special surface receptors that recognize IgG, called Fc receptors. When a particle is coated in IgG, these receptors bind to it, helping the macrophage grab onto it and pull it inside through phagocytosis. Clean, uncoated particles don’t have this tag, so the macrophage doesn’t recognize them as easily and is less likely to take them up. Which leads to IgG-coated particles being phagocytosed more often and more efficiently than clean ones.