Quantitative Flow Cytometry to Study Labeled Protein-Phospholipid Vesicle Interactions

Published: June 29, 2023

Abstract

Source: Podoplelova, N. et al., Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry. J. Vis. Exp. (2022)

In this video, we demonstrate the interaction between proteins and artificial phospholipid vesicles using quantitative flow cytometry. The binding of fluorescently-labeled proteins to fluorescently-labeled phospholipid vesicles increases the mean fluorescence intensity, and this increase is detected by a flow cytometer.

Protocol

1. Detection of protein-lipid interaction by flow cytometry Kinetic binding experiments Dilute phospholipid vesicles in Tyrode's buffer (20 mM HEPES, 150 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.4 mM NaH2PO4, 2.5 mM CaCl2, 5 mM glucose, 0.5% BSA, pH 7.4) to a concentration of 1 µM and total volume of 250 µL. Mix fluorescent-labeled coagulation factor X (fX-fd) from step 1 at a concentration of 500 nM with the phospholi…

Divulgazioni

The authors have nothing to disclose.

Materials

Alexa Fluor 647 NHS Ester (Succinimidyl Ester) Thermo Fisher Scientific A37573 Fluorescent dye
BD FACSCantoII BD Bioscience
DiIC16(3) (1,1'-Dihexadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate) Thermo Fisher Scientific D384 Fluorescent dye
DMSO Sigma Aldrich D8418
HEPES Sigma Aldrich H4034-500G
L-α-phosphatidylserine (Brain, Porcine) (sodium salt) Avanti Polar Lipids 840032P
L-α-phosphatidylcholine (Brain, Porcine) Avanti Polar Lipids 840053P
Mini-Extruder Avanti Polar Lipids 610020-1EA
Potassium chloride Sigma Aldrich P9541-500G
Calcium chloride, anhydrous, powder, ≥97% Sigma Aldrich C4901-100G
Sodium phosphate monobasic Sigma Aldrich S3139-250G
Sodium chloride Sigma Aldrich S3014-500G
Magnesium chloride Sigma Aldrich M8266-100G
Bovine serum albumin VWR Life Science AMRESCO Am-O332-0.1
EDTA disodium salt VWR Life Science AMRESCO Am-O105B-0.1

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Citazione di questo articolo
Quantitative Flow Cytometry to Study Labeled Protein-Phospholipid Vesicle Interactions. J. Vis. Exp. (Pending Publication), e21442, doi: (2023).

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