A FRET Flow Cytometry Technique to Detect Tau-Seed Induced Reporter Protein Aggregation
Published: March 29, 2024
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…
Açıklamalar
The authors have nothing to disclose.
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 |
Etiketler
Bu Makaleden Alıntı Yapın
A FRET Flow Cytometry Technique to Detect Tau-Seed Induced Reporter Protein Aggregation. J. Vis. Exp. (Pending Publication), e22070, doi: (2024).