Cell bio - design an experiment (PIKfyve)

Question

1) You found an inhibitor for PIKfyve. Design an experiment to demonstrate that PIKfyve is required for macrophage phagocytosis of IgG-opsonized red blood cells.

 

2) if you design a properly CONTROLLED experiment. You should include both a positive and a negative control.

Answer 1

This was a very difficult question to answer maybe because it was so lengthy! But better late than never. PIKfyve is an enzyme that plays a significant role in cellular processes. The name "PIKfyve" stands for "Phosphoinositide Kinase, FYVE-type Zinc Finger-containing.” [1/2].

  

Here’s an example of a possible experimental design:

 

Objective of this experiment:

 

To assess the role of PIKfyve in the phagocytosis of IgG-opsonized red blood cells by macrophages.

 

Experimental Design

Cell Culture: Grow macrophage cells in suitable culture conditions.

Prepare IgG-opsonized red blood cells (RBCs).

 

Treatment Groups

 

Experimental Group: Treat macrophages with the PIKfyve inhibitor.

 

Positive Control Group: Macrophages with no inhibitor treatment (to ensure they can perform phagocytosis under normal conditions).

 

Negative Control Group: Macrophages treated with a known phagocytosis inhibitor (to confirm the inhibition of phagocytosis).

 

Phagocytosis Assay:

 

1. Expose all groups of macrophages to IgG-opsonized RBCs for a set period (e.g., 1 hour).

2. Wash the cultures to remove non-phagocytosed RBCs.

3. Use a suitable method to quantify phagocytosis, such as microscopy to count engulfed RBCs or flow cytometry if the RBCs are fluorescently labeled.

 

Assessment: Compare the number of phagocytosed RBCs in the experimental group with both control groups.

 

A significant reduction in phagocytosis in the experimental group compared to the positive control would suggest that PIKfyve is required for macrophage phagocytosis of IgG-opsonized RBCs. The negative control confirms the assay's ability to detect inhibited phagocytosis.

 

Statistical Analysis (Fun part)

 

Perform statistical tests (e.g., ANOVA followed by post-hoc tests) to determine if the differences are significant.

 

Things To Take Into Considerations

 

Dose-Response Experiment: To strengthen the results, include different concentrations of the PIKfyve inhibitor in separate groups.

 

Time-Point Analysis: Assess phagocytosis at multiple time points to understand the temporal effects of PIKfyve inhibition.

 

Specificity of Inhibitor: Ensure the inhibitor specifically targets PIKfyve to rule out off-target effects.

 

Cell Viability: Check macrophage viability in all groups to ensure that observed effects are due to inhibition of phagocytosis, not cell death.

 

 

Reference:

 

1. PIKFYVE phosphoinositide kinase, FYVE-type zinc finger containing [Homo sapiens (human)]." National Center for Biotechnology Information. Gene ID: 200576, updated on 23-Nov-2023. Available at: https://www.ncbi.nlm.nih.gov/gene/200576

 

2. Rivero-Ríos P, Weisman LS. Roles of PIKfyve in multiple cellular pathways. Curr Opin Cell Biol. 2022 Jun;76:102086. doi: 10.1016/j.ceb.2022.102086. Epub 2022 May 16. PMID: 35584589; PMCID: PMC9108489.

 

3. Ali Z, Bhaskar SB. Basic statistical tools in research and data analysis. Indian J Anaesth. 2016 Sep;60(9):662-669. doi: 10.4103/0019-5049.190623. Erratum in: Indian J Anaesth. 2016 Oct;60(10 ):790. PMID: 27729694; PMCID: PMC5037948.

4.Holland-Letz T, Kopp-Schneider A. Optimal experimental designs for dose-response studies with continuous endpoints. Arch Toxicol. 2015 Nov;89(11):2059-68. doi: 10.1007/s00204-014-1335-2. Epub 2014 Aug 26. PMID: 25155192; PMCID: PMC4655015.