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).