Home › Cell Biology › siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells
Cell Biology JoVE (Open Access) Citable · DOI

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

DOI: 10.3791/60190-v
What you'll learn
  • ✓ Generate immature and mature monocyte-derived dendritic cells from human blood
  • ✓ Infect dendritic cells with HSV-1 and modulate autophagic flux using inhibitors
  • ✓ Perform siRNA electroporation to knock down FIP200 and assess autophagy
  • ✓ Analyze autophagic flux changes in infected dendritic cells via flow cytometry
Protocol

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

Difficulty
advanced
Total time
~7-10 days (cell differentiation 5-7 days, viral infection and analysis 1-2 days)
Model organism
Human monocyte-derived dendritic cells (in vitro)
Biosafety
BSL-2

Steps

1
Generate and culture immature and mature dendritic cells

Isolate monocytes from human blood and differentiate into immature dendritic cells (iDCs) using cytokine stimulation, then mature iDCs into mDCs. This foundational step establishes the primary cell populations for downstream HSV-1 infection experiments.

▶ 01:02
2
Infect dendritic cells with HSV-1 and apply autophagy inhibitors

Expose differentiated dendritic cells to HSV-1 and simultaneously treat with pharmacological inhibitors (Spautin-1 or Bafilomycin-A1) to block autophagic flux. Monitor infection kinetics and autophagy response in parallel samples.

▶ 02:02
3
Electroporat iDCs with FIP200-targeted siRNA

Perform electroporation of immature dendritic cells using FIP200-specific siRNA to achieve gene silencing. This technique depletes a key autophagy regulator before HSV-1 infection to assess its role in autophagic flux.

▶ 04:17
4
Analyze autophagic flux in infected dendritic cell populations

Assess autophagic flux changes in HSV-1-infected iDCs and mDCs using flow cytometry and autophagy markers (e.g., LC3-II, p62). Compare effects of pharmacological inhibition versus siRNA-mediated knockdown on autophagy dynamics.

▶ 06:21
💬 Comments coming soon