Bms11100 Advanced Immunology Assessment Answer

Question:

Experiment set up

1. Collect a sterile universal tube containing THP-1 monocyte cells, at 2 x 10⁶ cellsml^-1, from the 37^oC incubator and transfer to a sterile Class II hood.

2. Gently mix the tube to re-suspend the THP-1 cells and using a sterile pipette transfer 1ml of THP-1 cell suspension to each well of the sterile 6-well plate.

3. Using a sterile pipette, transfer 1ml of the treatments (RPMI medium, LPS, AA nZnO, ND nZnO, ZnSO₄ or bZnO) to the wells of the 6-well plate as shown below. There should be a final volume of 2ml in each well and a final THP-1 monocyte cell concentration of 1 x 10⁶ cells ml^-1

4. Incubate for 3h at 37^oC.

5. Before leaving the lab, prepare TWO appropriately-labelled Eppendorf tubes to collect 200ml of treated THP-1 cell suspension (Dr Proudfoot will instruct which two will be taken) after the 3h incubation period.

In addition, prepare six labelled Eppendorf tubes to help the module team to remove the supernatants 24h later to be stored in the -20^oC freezer for the ELISA.

6. After 3h incubation, remove the 6-well plate from the incubator in 1.C.07 and carefully transfer it to the sterile culture cabinet where 200ml should be removed from each well into an appropriately-labelled Eppendorf tube (prepared previously). Return the 6-well plate to the incubator so that it can be harvested by the module team the following day (supernatants will be frozen and stored for the IL-8 ELISA measurement next week).

7.  Carefully transfer your TWO eppendorf tubes (in an appropriate holder) up the stairs to lab 3.C.20 (N.B. carry the holder with a gloved hand while the other hand has no glove to be able to touch door handles safely).

8. It is now safe to prepare the THP-1 cells in a non-sterile environment but work over the absorbent sheet and wear gloves.

9. Carry out a cytospin and Diff-Quik stain (see separate protocol) on the selected samples. Observe at x400 magnification.

Related information and questions

· Zinc oxide nanoparticles (nZnO, 70nm), zinc oxide bulk particles (bZnO, >400mm) (Alfa Aesar, UK) and ZnSO₄ are supplied at a concentration of 300mM in RPMI medium. What will the final concentration be in the well?

· Lipopolysaccharide (from Escherichia coli) is supplied at a concentration of  200ngml^-1 in RPMI medium. What will the final concentration be in the well?

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