To verify the gene editing efficacies in the single-copy EGFPΔfluor and EGFPY66S reporter HAP1 cell lines, FACS analyses were performed 48 h after iTOP transduction. Cells in each well were trypsinized and resuspended in 200 μl of FACS buffer (5% FBS in 1 × DPBS) containing 1:1000 DAPI (4′,6-diamidino-2-phenylindole) DNA dye (Sigma). For the beta-2-microglobulin (B2M)-deficient HAP1 cells, 48 h after iTOP transduction, cells from each well were firstly trypsinized and then incubated in 50 μl of staining solution (1% FITC-conjugated anti-human HLA-A, B, C antibody (Biolegend) in FACS buffer) for 10 min at 4°C. After washing three times with 1× DPBS, cells were resuspended in 150 μl FACS buffer containing 1:1000 DAPI DNA dye. FACS analyses were carried out on a CytoFLEX LX system (Beckman). In all experiments, the total number of 10 000 events were acquired and were gated based on side and forward light-scatter parameters. Constitutive EGFP-expressing control HAP1 cells were used to adjust the parameters for the identification and gating of EGFP/FITC positive cells. The EGFP/FITC signal was detected using the 488 nm diode laser for excitation and the 525/40 nm filter for emission.
Cytoflex lx system
Manufactured by Beckman Coulter
Sourced in United States
The CytoFLEX LX system is a flow cytometry instrument designed for multi-parameter analysis of cells and particles. It features a modular design and flexible configuration options to accommodate a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Cytexpert software, by Beckman Coulter (2 mentions)
True stain monocyte blocker, by BioLegend (1 mentions)
Lymphoprep density gradient, by STEMCELL (1 mentions)
Brilliant stain buffer, by BD (1 mentions)
E607008 0500, by Sangon (1 mentions)
Collagenase solution, by Roche (1 mentions)
Staining buffer, by BioLegend (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cytexpert 2, by Beckman Coulter (1 mentions)
Diva 8, by BD (1 mentions)
Cd206 apc, by Thermo Fisher Scientific (1 mentions)
Cd8 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd45 efluor450, by Thermo Fisher Scientific (1 mentions)
Cd11b apc cy7, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc, by Thermo Fisher Scientific (1 mentions)
F4 80 pe, by Thermo Fisher Scientific (1 mentions)
Legendplex software v8, by BioLegend (1 mentions)
Legendplex mouse proinflammatory chemokine panel, by BioLegend (1 mentions)
Senescence β galactosidase staining kit, by Beyotime (1 mentions)
13 protocols using cytoflex lx system
1
Evaluating EGFP Knockout Efficiency
2
Multiparametric Phenotyping of PBMCs
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PBMCs were isolated from peripheral blood using a Lymphoprep density gradient (STEMCELL Technologies, Vancouver, Canada). Each sample received a cocktail containing 10 μL Brilliant Stain Buffer (BD Biosciences, Franklin Lakes, NJ, USA), 5 μL True-Stain Monocyte Blocker (BioLegend, San Diego, CA, USA), and the following surface marker antibodies: anti-CD19 (PE-Dazzle594), anti-CD3 (APC), anti-CD16 (Alexa700), HLA-DR (APC/Fire750), and anti-CTLA-4 (PE-Cy7). The following antibodies were then added to each tube individually: anti-CD8 (BUV496), anti-CD4 (BUV661), anti-CD45 (BUV805), anti-CD103 (BV421), anti-TIM3 (BV605), anti CD56 (BV650), anti-LAG-3 (BV711), anti-CD14 (BB785), and anti-PD-1 (BB700), before incubating at room temperature for 30 min. Cells were fixed and permeabilized in 1× incellMAX (IncellDx, San Carlos, CA, USA) for 60 min at room temperature in the dark. Cells were washed once with 2% bovine serum albumin (BSA) solution, and analyzed using a Cytoflex LX system with 355 nm (20 mW), 405 nm (80 mW), 488 nm (50 mW), 561 nm (30 mW), 638 nm (50 mW), and 808 nm (60 mW) lasers (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Analysis was performed with Kaluza version 2.1 software. The panel used in this study is shown in Supplementary Table 1.
3
Murine Th1/Th2 Cell Profiling by Flow Cytometry
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The prevalence of Th1, Th2 cells in spleen cells was detected by flow cytometry using Mouse Th1/Th2 staining kit. In brief, spleen tissues were homogenized in PBS balance solution. The homogenate was incubated in RPMI 1640 cell culture medium containing 10% fetal bovine serum, with 1 µL PMA/Ionomycin Mixture (250×) and 1 µL BFA/Monensin Mixture (250×), mixed, and cultured for 5 h at 37 ℃. Cells were stained by fluorescein isothiocyanate (FITC) anti-CD3, peridinin-chlorophyll-protein complex (PerCP)-cyanine (Cy) 5.5 anti-CD4, R-phycoerythrin (PE) anti-IFN-γ, and Allophycocyanin (APC) anti-IL-4. Measurements were performed using the CytoFLEX LX system, and data were analyzed with CytExpert Software (Beckman Coulter). Cell events were gated using forward-scatter and side-scatter plots. Then, CD3+ CD4+ cells were gated based on FITC and PC5.5 fluorescence intensity before Th1/Th2 cells were gated from the pool of CD3+ CD4+ cells according to IL-4 and IFN-g level (APC and PE fluorescence intensity, respectively).
4
Flow Cytometry Analysis of BMSC Markers
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BMSCs in passage 3 were used for flow cytometry analysis. Flow cytometry analysis of BMSC surface markers was performed as follows. BMSCs were suspended in phosphate-buffered saline (PBS; E607008-0500, Sangon Biotech, China) at a final concentration of 1 × 106/mL. Then, monoclonal antibodies and the isotype control were added to 100 μL cell suspensions and incubated for 60 min at 4 °C. The cell suspensions were centrifuged at 2000 rpm for 3 min to remove the antibodies and washed with PBS 3 times. Finally, 500 μL PBS was used to resuspend the cell pellet, which was analyzed with the CytoFLEX LX system (Beckman Coulter, USA). FlowJo software (Leonard Herzenberg Laboratory, USA) was used to analyze flow cytometry data.
5
Endothelial Cell and PTDC Staining
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For endothelial cell and PTDC staining, the residual scaffolds in PTs were first discarded, and then, the freshly obtained PTs were crushed in staining buffer (Biolegend) with a mortar and pestle. Single-cell suspensions were obtained with an additional digestion in collagenase solution (Roche Diagnostics) at 37 °C for 30 min.
Cell suspensions were then filtered with a 40 μm cell strainer and spun at 300 × g for 5 min at 4 °C. The supernatant was removed, and the deposit was resuspended in staining buffer. Then, the resuspended single cells were stained with an antibody cocktail for 60 min at 4 °C. After incubation, the cells were washed and resuspended in adequate staining buffer again and used for flow cytometric analysis.
Flow cytometric analyses were carried out using a CytoFlex LX system equipped with CytExpert 2.3 software (Beckman Coulter) or a Symphony A5 system equipped with Diva 8.0 software (BD Biosciences). Dead cells were excluded using a Live/Dead Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) or DAPI solution (1 μg·mL−1). We used fluorescence minus one control to define the boundaries between the positively and negatively stained cell populations. Flow cytometric data were analyzed with FlowJo V10 (Three Star) or CytExpert 2.3.
Cell suspensions were then filtered with a 40 μm cell strainer and spun at 300 × g for 5 min at 4 °C. The supernatant was removed, and the deposit was resuspended in staining buffer. Then, the resuspended single cells were stained with an antibody cocktail for 60 min at 4 °C. After incubation, the cells were washed and resuspended in adequate staining buffer again and used for flow cytometric analysis.
Flow cytometric analyses were carried out using a CytoFlex LX system equipped with CytExpert 2.3 software (Beckman Coulter) or a Symphony A5 system equipped with Diva 8.0 software (BD Biosciences). Dead cells were excluded using a Live/Dead Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) or DAPI solution (1 μg·mL−1). We used fluorescence minus one control to define the boundaries between the positively and negatively stained cell populations. Flow cytometric data were analyzed with FlowJo V10 (Three Star) or CytExpert 2.3.
6
Tumor Immune Cell Profiling Protocol
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The following antibodies were used for the analysis of tumor infiltration by immune cells: CD45 -eFluor 450 (eBioscience 48-0451-82), CD11b-APC cy7 (eBioscience 47-0112-82), F4/80-PE (eBioscience 12-4801-82), CD8-PE cy7 (eBioscience 25-0081-82), CD4-FITC (eBioscience 11-0042-85), IFNgama-APC (eBioscience 17-7311-82), CD206-APC (eBioscience 17-2061-82). Single-cell suspensions were prepared from the tumor tissues after homogenization or from culture suspension cells. For staining, 100 μl of single-cell suspensions were incubated with antibodies for 30 min. Individual single-color controls were prepared for compensation adjustment. After staining, the samples were washed twice with PBS and resuspended in 600 μl PBS. Flow cytometry data were acquired using a CytoFLEX LX system (Beckman Coulter). FlowJo version 10.0 was used for data analysis. The absolute numbers of CD8+ T cell or macrophage numbers per gram of tumor were the corresponding cell counts from 60 μl of cell suspension (10% of total volume) divided by the tumor weight. The gating strategy is described in each figure’s caption. An example of the gating strategy is given in Supplementary Figure S1 .
7
Characterizing hAEC Markers and Apoptosis
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FACS of hAEC markers and apoptotic cells was performed using flow cytometry. The cultured hAECs were digested into single cells with 0.25% trypsin-EDTA and resuspended in PBS at a final concentration of 5 × 105 cells/mL. For the analysis of hAEC markers, primary antibodies (CD29, CD73, CD326, HLA-ABC and isotype control) were added to 100 μL of a cell suspension and incubated for 1 h at 4°C. The cells were washed twice with 2 mL PBS and centrifuged at 500 × g for 5 min. For the analysis of SSEA4, the cells were fixed with 4% PFA for 5 min, permeabilized with 0.3% Triton X-100, blocked with 3% BSA for 1 h, and then sequentially incubated with the primary antibody and secondary antibody. Five hundred microliters of PBS were added to resuspend the cell pellets. For the cell apoptosis assay, hAECs were digested with 0.25% trypsin and stained in 100 μL binding buffer with 5 μL of Annexin V-Alexa Fluor 488 and 10 μL of PI Staining Solution (20 μg/mL) according to the manufacturer’s instruction (40305ES20, Yeasen Biotechnology, China). All cells were analyzed using the CytoFLEX LX system (Beckman Coulter, USA). The results were evaluated using FlowJo software (Leonard Herzenberg Laboratory, USA).
8
Flow Cytometry Analysis of Cells
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Cells were prepared for flow cytometry analysis as described (Baum et al. 2004 (link); Hirschmann et al. 2014 (link)). Propidium iodide fluorescence was detected in a Beckman Coulter CytoFlex LX system with a 675/30 nm filter. Data were analyzed using CytExpert software (Beckman Coulter), and representative graphs were plotted manually using values obtained from the software.
9
Annexin V and PI Apoptosis Assay
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A YF 647A-Annexin V and PI Apoptosis Kit (Yuheng Biotechnology Co., Ltd., Suzhou, China) was used for flow cytometry. First, the medium was gathered in a 15 ml centrifuge tube. Then, the cells were digested in trypsin-free EDTA, centrifuged with the retained medium at 300 g for 5 min at 4°C, and the supernatant was discarded. Next, the cells were resuspended and centrifuged twice with PBS under the same conditions. The supernatant was then got rid of and the cells were gently resuspended in 100 μl of binding buffer. Then, 5 μl of YF647A-Annexin V and 5 μl of PI were mixed to each tube and incubated at 24°C in the dark for 15 min. At last, the experiment was detected with a CytoFLEX LX system (Beckman Coulter, United States); data were analyzed with CytExpert software.
10
Quantifying CasRx Editing Efficiency
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CasRx-engineered cells were transduced with the pLenti-EGFPdestablized-Hygro (Addgene, cat. no. 138152 (ref. 43 (link))) lentivirus and selected with hygromycin for 1 week. After selection, the cell culture was split into two different dishes. One of the dishes was transduced with the pLKO5-CasRx(DR1 30)–EFS-tRFP657 containing a NT gRNA and the other with a GPF-targeting gRNA. Four days after transduction, the cells were resuspended in FACS buffer (PBS, 3% FBS, 5 mM EDTA) and recorded using a Beckman Coulter Cytoflex LX system. FlowJo v.10.7.2 was used to analyze populations. CasRx activity was calculated as one minus the ratio between the GFP intensity (mean B525-FITC-A) of cells transduced with the gGFP gRNA and cells transduced with the gNT gRNA. The pLKO5-CasRx(DR1 30)–EFS-tRFP657 vector was cloned using the pLKO5.sgRNA.EFS.tRFP (Addgene cat. no. 57823 (ref. 83 (link))).
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