The study of various cellular life processes has been made easier by the detection of intermolecular interactions in vivo and in vitro through a variety of methods. A very valuable tool used to study the intermolecular interactions associated with dynamic life processes in living cells is the utilization of the fluorescence resonance energy transfer (FRET) phenomenon coupled with the use of selective fluorescein-coupled target molecules. Many reports have utilized fluorophore-coupled antibodies for FRET analysis. However, these methods are limited to extracellular molecules and suitable antibodies must be available. Currently, the development of green fluorescent protein (GFP) variants suitable for FRET has expanded the use of this method, not only for extracellular but also for intracellular molecular processes, and the combination of FRET and flow cytometric techniques has allowed the study of intermolecular interactions to be carried out in a high-throughput and efficient manner.
Principle
Flow cytometric analysis of fluorescence resonance energy transfer is based on the principle of constructing fusion proteins of GFP variants and transferring them into an appropriate cell system that can be used to monitor the molecular interactions of different biological mechanisms in living cells by flow cytometry.The use of FRET analysis in combination with flow cytometry will allow researchers to screen a large number of cells in a short period of time.
Operation method
Flow cytometric analysis of fluorescence resonance energy transfer
Principle
Flow cytometric analysis of fluorescence resonance energy transfer is based on the principle of constructing fusion proteins of GFP variants and transferring them into an appropriate cell system that can be used to monitor the molecular interactions of different biological mechanisms in living cells by flow cytometry.The use of FRET analysis in combination with flow cytometry will allow researchers to screen a large number of cells in a short period of time.
Materials and Instruments
Equipment: Move The basic process of flow cytometric analysis of fluorescence resonance energy transfer can be divided into the following steps: 1.1 Construction of Vectors and CFP and YFP Carriers 1.1.1 Design PCR primers for insertion of CFP and YFP cDNA. 1.1.2 PCR with pECFP-N1 or pEYFP-N1 template. 1.1.3 Separate the PCR products by electrophoresis on a 1% agarose gel. The correct fragments are excised. The correct fragments are excised and purified by QIAEX. 1.1.4 The PCR fragments are digested with the following endonucleases: (1) BamHI/EcoRV (YFP fragment). (2) EcoRV/Xbal (CFP and YFP fragments). 1.1.5 The pEF6-myc-HisB vector is digested with the following endonucleases: (1) BamHI/EcoRV (2) EcoRV/Xbal. 1.1.6 Fix the resulting fragment with the carrier from 5 a. Ligate the fragment to the vector to construct the pEF6B-YFP-C plasmid. The CFP or YFP fragments from step 4 b are also ligated to the vector from step 5 b to construct the pEF6B-CFP-N and pEF6B-CFP-N plasmids. 1.1.7 Transform the plasmids into XL-1-blue receptor bacteria. 1.1.8 The plasmids are prepared in small quantities and the resulting clones are screened for insertion of PCR products. 1.1.9 A small amount of DNA plasmid is prepared and sequenced to determine the integrity of the insert sequence. 1.2 cDNA cloning 1.2.1 PCR amplification of cDNAs for p80 TNFR-2, TRAF2 and SODD. 1.2.2 Amplification of p80 TNFR-2, TRAF2 and SODD by BamBam. 1.2.2 The p80 PCR product is digested with BamHI/EcoRV. The EcoRV/Xbal product is digested with BamHI/EcoRV. The TRAF2 and SODD PCR products were digested with EcoRV/Xbal. 1.2.3 The EcoRV/Xbal product was digested with EcoRV/Xbal. 1.2.3 Enzymatically digest pEF6B-YFP-C with EcoRV/Xbal. pEF6B-YFP-TRAF2 and pEF6B-YFP-SODD vectors are constructed by ligating and inserting the TRAF2 and SODD cDNAs of the products from step 2 into the pEF6B-YFP-TRAF2 and pEF6B-YFP-SODD vectors. 1.2.4 Enzymatically cleave pEF6B-CFP-N and pEF6B-YFP-N with BamHI/EcoRV. Insert the p80 fragments of the products from step 2 into the pEF6B-p80-CFP and pEF6B-p80-YFP vectors. 1.2.5 Repeat the above steps.
① 6 ml FACS sorting tube (Falcon).
② FACSVantage SE flow cytometer (BD Biosciences, San Jose, CA) (see Subheading 3.3.3.).
③ FlowJo analysis software (Tree Star, Inc., San Carlos, CA) or other flow cytometry analysis software.
④ Centrifuge
Reagents:
① Plasmids: pEF6-myc-HisB (Invitrogen, Carlsbad, CA), pECFP-N1, pEYFP-N1 (BD Biosci-ences Clontech, Palo Alto, CA).
② cDNAs for p80 TNFR-2, TRAF2, and SODD (Structural Silencer of the Ductus).
③ PCR oligonucleotide primers.
③ PCR oligonucleotide primers. ④ Restriction enzymes, T4DNA ligase.
⑤ QIAEX gel purification kit (Qiagen, Valencia, CA).
⑥ Active E. coli cells for transformation (e.g., XL-1 Blue-Stratagene, La Jolla, CA).
⑦ HEK 293T cells.
⑧ Complete Dulbecco's modified DMEM: DMEM without phenol red, 10% fetal calf serum (FCS), 100 units of penicillin and streptomycin, and 2 mM L-glutamine.
⑨ FuGENE 6 (Roche Applied Science, Indianapolis, Indiana).
⑩ Phosphate buffer solution (PBS).
⑪ Nonai detergent (NP-40): 10 mM Tris-HCl, pH 7.5,150 mM NaCI, and 1% NP-40.
⑫ Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA).
⑬ 10% Bis-Tris NuPA protein gel (Invitrogen).
⑭ Rabbit polyclonal anti-TRAF2 serum (Santa Cruz Biotechnology, Santa Cruz, CA).
⑮ 500 μg/ml PI solution.
2.1 Transfection in 293T cells
2.1.1 Inoculate 2.5 × 105 cells per well in a 12-well plate with 1 ml of complete DMEM medium and incubate at 37 ℃ for 16-20 hours.
2.1.2 Transfect 2 μg of each plasmid into HEK 293T cells with 6 μl FuGENE 6, the ratio of DNA/FuGENE 6 is 1:3.
2.1.3 Grow and incubate the cells at 37 ℃ for 24~48 h.
2.1.4 Harvest cells for subsequent Western blotting or flow-through analysis.
2.2 Western Blotting Analysis
2.2.1 Aspirate the culture flower from the wells and add 1 ml of PBS to each well. gently resuspend the cells in PBS and transfer the cell suspension to a centrifuge tube.
2.2.2 Centrifuge the cells at 480 g for 5 min. remove the PBS.
2.2.3 Resuspend cells in 100 μl NP-40 lysate and incubate on ice for 15 min.
2.2.4 Centrifuge the cells at 17 900 g for 10 min at 4 ℃.
2.2.5 Transfer the supernatant to a fresh centrifuge tube. Measure the protein concentration with Bio-Rad Protein Assay Kit.
2.2.6 Add 50 μg of lysed protein extract to a 10% Bis-Tris NuPAGE gel and transfer to a nitrocellulose membrane.
2.2.7 Use anti-TRAF2 antibody and HRP-coupled hybridized anti-rabbit IgG secondary antibody. Figure 15-2 shows the expression of YFP-TRAF2 (lane 2) and unlabeled TRAF2 (lane 3) in HEK 293T cells (see Note 4.4).
2.3 Monitoring the fluorescence of expressed proteins As an alternative to the Western blot assay, transfected plasmid-expressing cells can also be monitored by flow cytometry.
2.1 Wash the cells twice with 1 ml of PBS containing 2% FCS and filter by centrifugation at 480 g for 5 min at 4 ℃.
2.2 Resuspend cells in 1 ml of PBS containing 2% FCS.
2.3 Add 2 μl of PI solution (500 μg/ml) to the cell suspension.
2.4 Sample was analyzed by flow cytometry.
3 Perform FRET flow cytometry analysis1. Transfection
The transfection procedure was performed as described above using FuGENE6.
2. Cell Harvesting
2.1 24~48 h, wash the cells twice by centrifugation with PBS containing 2% FCS, and harvest the cells.
2.2 Resuspend cells in PBS containing 2% FCS.
2.3 The cells are filtered through a nylon sieve and analyzed.
2.4 Keep the cells at 4 °C until the assay is performed. If further manipulation of the cell samples is required (e.g., fitosome stimulation), they can be stored at room temperature.
3、Flow cytometry assay
Cells were analyzed on a FACS Vantage SE flow cytometer.The FACS Vantage SE uses dual lasers; an ILT air-cooled argon laser and a krypton laser equipped with violet light (Spectra-Physics model 2 060, Spectra-Physics, Mountain View, CA). The argon laser was tuned to 514 nm for direct excitation of the YFP, and the krypton laser was tuned to 513 nm for excitation of the CFP. Forward (FSC) and side-to-side (SSC) filters were replaced with 513/10 nm bandpass filters (BP), and CFP fluorescence was detected with a 470/20 nm BP filter at FL5 (P6).505 A long-pass beam-splitting prism (DM) was used to separate CFP fluorescence and FRET excitation from laser 2. CFP fluorescence is detected in FL5 with a 470/20 nm BP filter (P6).505 A long-pass beam splitter prism (DM) is used to separate CFP fluorescence from FRET excitation from Laser 2. FRET signals are detected in FL4 with a 546/10 nm BP filter (P5).505 The YFP fluorescence is detected in FL1 (P3) directly with a 546/10 nm BP filter but is directed to the P7 channel, which allows for the fluorescence compensation adjustments between P5 and P7 to be made with the Omnicomp option. The hydraulic pressure should be set at 30 pounds per square inch (psi) at the time of sample collection, which shortens the pulse timing between the argon and krypton lasers to allow for interlaser compensation adjustments with the standard delay mode, with a maximum delay time of 17.5 μs. 50,000 viable cells were collected by adding 1 mg/ml PI prior to assaying.
4 FlowJo Analysis Data4.1 Live cell gates were set via SSC/FSC or PI/FSC (detected in P8) scatter plots.
4.2 Create a 2D scatter plot of CFP (FL5/P6) fluorescence and FRET (FL4/P5) with live cell gates.
4.3 Set the cutoff value between CFP positive and CFP negative populations using vector-transfected samples as negative controls. This fixes the CFP-positive gate.
4.4 FRET fluorescence intensity was analyzed by histograms using cells within the CFP-positive gates.
4.5 Overlay FRET histograms using p80-CEP as a negative control. Bar graphs from Samples 6, 7 or 8 overlaid on Sample 2 bar graphs. Expression of p80-CFP and p80-YFP (Sample 6) produces a strong FRET signal. co-expression of p80-CFP and YFP-TRAF2 (Sample 7) also results in a strong FRET signal. However, when co-transfected with the control YFP-SODD, which does not interact with p80-CFP, no meaningful FRET signal could be generated that interacted with p80-CFP (Sample 8).
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