A FRET Flow Cytometry Technique to Detect Tau-Seed Induced Reporter Protein Aggregation

Abstract

Source: Furman, J. L., et al. Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry. J. Vis. Exp. (2015).

The video demonstrates a fluorescence resonance energy transfer (FRET) flow cytometry assay to detect the seeding activity of protein aggregates isolated from biological samples. Mammalian cells expressing tau reporter proteins are incubated with liposome transduction complexes containing tau seeds. These seeds mediate the aggregation of the reporter proteins, leading to generating a FRET positive signal in the flow cytometer.

Protocol

NOTE: This protocol emphasizes the use of FRET flow cytometry for detecting seeding activity from mouse biological samples. It is also compatible with recombinant fibrils and human biological samples. Mouse euthanasia and brain harvesting was performed in accordance with IACUC-approved procedures. 1. Brain Extraction Following deep anesthetization with isoflurane (2%), perfuse a mouse with ice-cold PBS containing 0.03% heparin, and extract the brai…

Representative Results

Table 1: Cell lines used with FRET flow cytometry. HEK 293T cells are used for flow cytometry setup. CFP single-positive cells are used for compensation (*). YFP single-positive cells are used to eliminate false FRET signal due to direct activation of YFP by 405 nm excitation. CFP/YFP dual-positive cells are FRET-compatible and serve as the biosensor cells. <img al…

Materials

TBS	Sigma	T5912
Complete Protease Inhibitors (EDTA-free)	Roche	4693159001
Cryo-vials	Sarstedt	72.694.006
Analytical Balance	Mettler Toledo	XSE 105DU
Weighing Boats	Fisher Scientific	13-735-743
15 mL conical tube	USA Scientific	1475-0501
Omni Sonic Ruptor Ultrasonic Homogenizer	Omni International	18-000-115
Micro-Tip for Ultrasonic Homogenizer	Omni International	OR-T-156
2-Propanol	Fisher Scientific	A451
Noise Cancelling Ear Muffs	Fisher Scientific	19-145-412
Kimwipes	Fisher Scientific	S47299
1.5 mL tubes	USA Scientific	1615-5510
Microcentrifuge	Eppendorf	5424 000.215
DPBS	Life Technologies	14190-136
DMEM	Life Technologies	11965-084
Fetal Bovine Serum	HyClone	SH30071.03
Penicillin-Streptomycin	Life Technologies	15140-122
GlutaMax	Life Technologies	35050-061
Trypsin-EDTA	Life Technologies	25300-054
50 mL Conical Tubes	Phenix Research	SS-PH15
25 mL reagent resevoirs	VWR	41428-954
Multi channel pipet	Fisher Scientific	TI13-690-049
96 well flat bottom plates	Corning	3603
Opti-MEM	Life Technologies	31985-070
Lipofectamine 2000	Invitrogen	11668019
96 well round bottom plates	Corning	3788
16% Paraformaldehyde	Electron Microscopy Sciences	RT 15710
PBS	Sigma-Aldrich	P5493
EDTA	Sigma-Aldrich	ED2SS
HBSS	Life Technologies	14185-052
Sorvall ST 40 Centrifuge	Thermo Scientific	75004509
BIOLiner Swinging Bucket Rotor	Thermo Scientific	75003796
Hemacytometer	VWR	15170-172
MACSQuant VYB Flow Cytomter	Miltenyi Biotec	130-096-116
Chill 96 Rack	Miltenyi Biotec	130-094-459
Flow Jo analysis software	Flow Jo
20 uL pipet tips	Rainin	GPS-L10
200 uL pipet tips	Rainin	GPS-250
1 mL pipet tips	Rainin	GPS-1000
200 uL pipet tips	USA Scientific	1111-1800
5 mL serological pipett	Phenix Research	SPG-606180
10 mL serological pipett	Phenix Research	SPG-607180
25 mL Serological pipett	Phenix Research	SPG-760180