Lentiviral-Mediated Transduction of Human Neurons for Tau Protein Aggregation Analysis

Abstract

Source: Aulston, B., et al. An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons. 

This video demonstrates the process of transducing human neuronal cells with a lentivirus encoding a mutant human tau protein tagged with yellow fluorescent protein (YFP) to study protein aggregation. It outlines the steps from viral entry to mutant tau expression, culminating in the formation of visible cytoplasmic tau aggregates in the neurons.

Protocol

1. Preparation of Media and Reagents

1. Thaw the basement membrane matrix coating for culture plates at 4 °C (do not allow the basement membrane matrix to warm up, or it will solidify). Make 1 mL aliquots and store them at -20 °C or -70 °C.
2. Reconstitute basic fibroblast growth factor (bFGF) in sterile phosphate-buffered saline (PBS) at 10 μg/mL and make 10 μL aliquots. Store them at 4 °C.
3. To a new, unopened, 500 mL bottle of DMEM/F12 with glutamine, add B27 (10 mL), N2 (5 mL), and penicillin-streptomycin (5 mL). Place 50 mL of this neural stem cell (NSC) media in a conical tube and add 10 _....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cm culture dishes	Thermofisher	12556002
50 mL tubes	Biopioneer	CNT-50
70% ethanol in spray bottle	Various sources	NA
B27 supplement	Thermofisher	17504044
Basement membrane matrix (Matrigel)	Corning	356231
Basic FGF	Biopioneer	HRP-0011
Bovine serum albumin	Sigma	A7906
Cell culture incubator	Various sources	NA
Centrifuge	Various sources	NA
DMEM-F12 culture media with glutamine	Thermofisher	10565042
Ethanol (50% concentration or higher)	Various sources	NA
Human neural stem cells	Various sources	NA
Lentiviral vectors	Various sources	custom order
N2 supplement	Thermofisher	17502048
Penicillin-Streptomycin	Thermofisher	15140122
Phosphate buffered saline	Thermofisher	14190250
Sterile cell culture hood	Various sources	NA
Water bath	Various sources	NA