Quantitative Flow Cytometry to Study Labeled Protein-Phospholipid Vesicle Interactions

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

1. Kinetic binding experiments
   1. 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.
   2. Mix fluorescent-labeled coagulation factor X (fX-fd) from step 1 at a concentration of 500 nM with the phospholipid vesicles from step 1.1.1 in a 1:1 ratio (final vesicle concentration 0.5 µM, fX-fd concentration is 250 nM of fX) to a total volume of 500 µL.
   3. Immediately inject the 500 µL of the mixed suspension (~20 min for analysis with a low flowing rate) into the flow cytometer. Use a low flow rate and ensure that the threshold for channel FL2 (excitation 488 nm, emission filter 585/42 nm) is as value 200. Measure the mean fluorescence in channel FL4 (excitation 633 nm, emission filter 660/20) for the fluorescence dye from the Table of Materials.
      NOTE: Choose a cytometer without an autosampler. This will speed up the process of injection of the sample into the measuring cell.
   4. When saturation of binding is achieved (no significant increase in fluorescence within 5 min), rapidly dilute the sample 20-fold with Tyrode's buffer, and monitor the dissociation until baseline fluorescence is reached (complete dissociation) or until a plateau is reached (no significant decrease in fluorescence within 5 min).
      NOTE: As a control, add 10 µM EDTA and monitor complete dissociation for 5 min.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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