THP1-Difluo™ hLC3 Cells

Monitoring autophagy activity 

THP1-Difluo™ hLC3 reporter cells are designed to monitor the autophagic flux by means of a fluorescent-labeled LC3B protein.

These cells, derived from the THP-1 human monocytic cell line, express the RFP::GFP::LC3 fusion protein in which the human LC3B (microtubule-associated protein 1 light chain 3 beta) is fused to two fluorescent reporter proteins: RFP (acid-stable) and GFP (acid-sensitive). The LC3B protein is a good marker to assess the autophagic flux since it is recruited from the cytosol to be associated to all the autophagic structures: the nascent autophagosome (called the isolation membrane), the complete autophagosome and the autolysosome. Upon autophagy induction, diffuse cytoplasmic fluorescence shifts to autophagosomes and autolysosomes as fluorescent puncta. 

Monitoring of autophagy activity relies on microscopy visualization of fluorescent puncta, progressive degradation of the GFP, and concurrent increase of RFP signal in autolysosomes. The number of fluorescent punctate structures per cell, the percentages of RFP-GFP positive and RFP positive cells can be used to assess autophagic flux as described previously [1,2]. 

THP1-Difluo™ hLC3 cells are resistant to Zeocin®.

Read our review on Autophagy and innate immunity.

References

1. Loos B. et al. 2014. Defining and measuring autophagosome flux- concept and reality. Autophagy. 2014;10(11):2087-96. 
2. Kimura S. et al., 2007. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent- tagged LC3. Autophagy. 3(5):452-60.

Figures

Confocal microscopy images of rapamycin-induced autophagosomes in THP1-Difluo™ hLC3 cells.
Cells were treated with phorbol myristate acetate (PMA; 50 ng/ml for 3 hours) to induce cell differentiation. After 3 days, cells were treated with rapamycin (25 μM for 6 hours) a commonly used autophagy inducer. Rapamycin treatment was performed in the presence of the autophagy inhibitor bafilomycin A1 (500 nM) to block the autophagic flux and allow visualization of discrete autophagic puncta. Cells were fixed with paraformaldehyde (PFA) prior to confocal imaging. Autophagosomes were visualized in the treated cells as both green (GFP) and red (RFP) puncta (appearing yellow in the merged image).

Specifications

Antibiotic resistance: Zeocin®

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum (FBS; 30 min at 56°C), 100 μg/ml Normocin™, Pen-Strep (100 U/ml-100 μg/ml).

Quality control:

• THP1-Difluo™ hLC3 cells have been tested for their ability to respond to autophagic inducers.
• The stability of this cell line for 20 passages following thawing has been verified.
• THP1-Difluo™ hLC3 cells are guaranteed mycoplasma-free.

This product is covered by a Limited Use License (See Terms and Conditions).

Contents

• 1 vial of THP1-Difluo™ hLC3 Cells (3-7 x 10⁶ cells)
• 1 ml of Zeocin® (100 mg/ml).
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.

 Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

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