For all staining, cells were blocked with 10% FBS prior to extracellular staining, then, if intracellularly stained, cells were treated with Permeabilization Solution (BD) and stained for intracellular markers in Perm/Wash Buffer (BD). Stains: Hoechst, and antibodies against CD45 (BioLegend, HI30; Tonbo, HI30), CD34 (BioLegend, 581), CD66b (BioLegend, G10F5), pan-Cytokeratin (pCK, Abcam, C-11), SIRT2 (Proteintech, 66410), Anti-histone H3-acetyl K18 (Abcam, 1191), p300 (Santa Cruz, 32244 AF488), acetyl-p300 (Invitrogen, PA5-16185).
Permeabilization solution
Manufactured by BD
Sourced in United States
Permeabilization solution is a laboratory reagent used to temporarily disrupt the integrity of cell membranes, allowing for the introduction of various molecules or compounds into the cells. The solution facilitates access to the cellular interior, enabling various experimental and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (4 mentions)
Fixation buffer, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Perm wash buffer, by BD (2 mentions)
Collagenase 1, by Merck Group (2 mentions)
Facsdiva software, by BD (2 mentions)
Sirt2, by Proteintech (1 mentions)
Anti histone h3 acetyl k18, by Abcam (1 mentions)
Facsalibur, by BD (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Apc anti mouse cd206, by BioLegend (1 mentions)
Apc anti human cd206, by BioLegend (1 mentions)
Fitc anti human cd14, by BioLegend (1 mentions)
Aria 3 cell sorter, by BD (1 mentions)
Pe anti mouse cd11b, by BioLegend (1 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysis solution, by BD (1 mentions)
Anti foxp3 apc, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
Mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions)
22 protocols using permeabilization solution
1
Multiparametric Immune Profiling
2
Quantification of IP-10 Expression in Blood Samples
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All antibodies were purchased from Biolegend (Pacific Heights Blvd, USA). Surface and intracellular staining was performed using previously described protocols (Xu et al., 2013 (link)). For CD14 surface staining, fresh heparinized peripheral blood (300 μl) was incubated with the CD14-APC antibody at room temperature for 20 min, after which 2 ml of haemolysin (BD Biosciences, USA) were added. The cells were incubated at room temperature for 5 min and centrifuged at 1,200 rpm for 5 min, after which the supernatant was discarded. For intracellular staining, 300 μl of permeabilization solution (BD Biosciences, USA) were added, incubated at room temperature for 30 min, and then 1 ml of 1× wash buffer was added. After centrifugation at 1,200 rpm for 5 min, 10 μl of IP-10-phycoerythrin (IP-10-PE) was added, and the cells were incubated at room temperature for 20 min. IP-10 expression was detected in vitro by centrifuging the cells, resuspending them in 1× Annexin V buffer, and then successively incubating them with 5 μl of Annexin V for 15 min at room temperature and with 10 μl of 7-AAD for 5 min at room temperature. Cells were pelleted and analysed using the FACSCalibur system and CELL Quest software to assess apoptosis. Corresponding isotype controls were added according to the manufacturer’s instructions.
3
Dissociation and FACS Analysis of hiPSC-CMs
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The cell samples were dissociated as previously reported [29 (link)]. The hiPSC-CMs were digested with collagenase I (1 mg/ml Sigma) with DNase I (40 Unit/ml Sigma) in PBS for 20 min, followed by 0.025% trypsin/EDTA treatment for 5 min at 37 °C. For analysis of cTnT- and Nkx2.5-positive cardiomyocytes from differentiated hiPSCs, cells were fixed and permeabilized with fixation a permeabilization solution for 30 min (BD bioscience) and stained with the primary anti-cardiac tropoinT antibodies or isotype control antibodies for 1 h, washed and stained with appropriate secondary antibodies. After staining, the cells were washed, resuspended, and evaluated with FACSalibur (BD Biosciences) and CELLQuest software.
4
Multiparametric Flow Cytometric Analysis of Immune Cell Phenotypes
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For surface staining, cells were washed and incubated with FITC-anti-human CD14 (BioLegend), PE-anti-human CD197 (BioLegend), APC-anti-human CD206 (BioLegend), or PerCP-5.5-anti-mouse F4/80 (BioLegend), PE-anti-mouse CD11b (BioLegend), APC-anti-mouse CD206 (BioLegend) in PBS containing 2% FBS at 4°C for 30 min. Isotype-matched immunoglobulin served as controls. For intracellular staining, cells were fixed with fixation buffer (BD Biosciences) for 30 min at 4°C. The fixed cells were permeabilized with the permeabilization solution (BD Biosciences) at room temperature for 30 min. Cells were incubated overnight with Alexa Fluor 488-anti-mouse iNOS (eBioscience) at 4°C. Then, labeled cells were sorted by using the Aria III cell sorter (BD Biosciences) or analyzed by using BD FACSCanto II (BD Biosciences) with FACSDiva software.
5
Immune Cell Profiling by Flow Cytometry
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The percentage of helper (CD4+) and cytotoxic (CD8+) T lymphocytes, regulatory T cells (CD4+CD25+FoxP3+), NK killer cells (NKR-P1+), and monocytes and macrophages (CD68+) in blood, spleen, and tumor samples was determined by flow cytometry using anti-CD4-phycoerythrin (PE), anti-CD8-PE, anti-NKR-P1-FITC, anti-CD25-FITC (Biosource, Washington, USA), anti-Foxp3-APC (eBioscience, San Diego, CA), and anti-CD68 monoclonal antibodies labeled with a secondary antibody coupled to APC (Biosource, Washington, USA). Briefly, 30 μL of blood or homogenate of spleen or tumor was incubated with 5 μL of the monoclonal antibody (1:10 dilution) for 30 min. Subsequently, 200 μL of red blood cell lysis solution was added (BD Biosciences), incubated for 10 min, and washed with PBS. 200 μL of permeabilization solution was added (BD Biosciences), incubated for 10 min, washed, incubated with anti-Foxp3-APC for 30 min, and finally washed and fixed with 1% paraformaldehyde in PBS. Cells were analyzed by flow cytometry (FACSCalibur, BD Biosciences) using CellQuest Pro (BD Biosciences) and Flow Jo version 10.
6
Quantifying T Cell Subsets by Flow Cytometry
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The frequency of CD4+IFN-γ+IL-17− Th1, CD4+IFN-γ−IL-17+ Th17, CD4+IFN-γ+IL-17+ Th1/17 and CD4+CD25+Foxp3+ T cells in individual samples were determined by flow cytometry following intracellular staining with anti-cytokine antibodies. Briefly, the stimulated PBMCs were harvested and stained with allophycocyanin (APC)-labeled anti-CD4, fixed with the Perm/Fix solution, and permeabilized, followed by staining with fluorescein isothiocyanate (FITC)-labeled anti–IL-17 and PE-Cy7-labeled anti-IFN-γ (Becton Dickinson, San Diego, USA). Additional cells were stained in duplicate with PerCP-anti-CD4/FITC-anti-CD25 or isotype-matched controls (BD PharMingen, San Diego, USA) for 30 min, fixed, and permeabilized using the permeabilization solution (BD Biosciences), followed by intracellular staining with PE-anti-Poxp-3 (BD PharMingen, San Diego, USA). After being washed with PBS, these cells were analyzed on a FACSCalibur (BD Biosciences, San Jose, USA) and at least 20,000 events were analyzed by FlowJo software (v7.6.2).
7
Multiparametric Flow Cytometry Analysis of Immune Cells
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At tumor endpoint, animals were euthanized by carbon dioxide asphyxiation. Single cell suspension of cells was harvested from the spleen and tumor. Tumors were dissociated using a mouse tumor dissociation kit (Miltenyi Biotec, CA, USA). Red blood cells are lysed using 1X red blood cell lysis buffer (Thermo Fisher Scientific, CA, USA). Remaining cells are resuspended in cell staining buffer (BioLegend, CA, USA) at a concentration of 2 × 105–1 × 106 cells per 100 µl. Fc receptors are blocked using 10 µg/ml ChromPure of mouse IgG (Jackson ImmunoResearch Inc., PA, USA) per 106 cells in a 100 µl volume for 10 min on ice. Cells are washed with cell staining buffer and incubated with antibody mix (Table S1 ) for 15–20 min on ice in the dark. Cells are fixed using 200 µl Fixation buffer (Thermo Fisher Scientific, CA, USA) for 20 min at 4ºC in the dark. Cells are washed 2 times with 1X PermWash (BD Bioscience, CA, USA) and permeabilized using permeabilization solution (BD Bioscience, CA, USA). Intracellular antibodies (Table S1 ) were incubated for 20 min at room temperature in the dark. Stained cells were analyzed using the Cytek® Aurora (Cytek, MD, USA) within 2 weeks of staining. Data were analyzed using FCS express (De Novo Software, CA, USA). Figure S12 shows a representative flow cytometry gating scheme identifying the various immune cell subsets.
8
Multicolor Flow Cytometry Analysis
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For surface staining, cells were incubated with fluorochrome- or biotin-labeled antibodies specific for mouse CD11c, PDCA-1, Siglec-H, B220, CD11b, CD4, CD8, CD44, CD19, CD21, CD23, CD86, I-Ad or H2-Kd (BD Pharmingen, Biolegend or eBioscience), followed by streptavidin in the case of biotinylated antibodies, as described [30 (link), 32 (link)]. Cells were then washed with PBS and fixed with 4% paraformadehyde. For intracellular staining, fixed cells were permeabilized with 2% saponin or permeabilization solution (BD, Pharmingen), and incubated with specific antibodies (anti-Foxp3; anti-LCMV-NP, VL-4). Surface and intracellularly stained cells were acquired on a BD FACSDiva-driven LSR II flow cytometer, and data were analyzed using FlowJo software (Treestar).
9
Th1/CD4+ T Cells and Apoptosis Analyses
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For Th1/CD4+ T cells percentage analysis, CD4+ T cells were collected and activated with PMA (50 ng/mL) for 2 h, and then monensin (3 μM, a transport inhibitor) was added for an additional 2-h incubation. After harvesting and washing with PBS, CD4+ T cells were permeabilized with permeabilization solution (BD Biosciences, USA) for 10 min and fixed with 4% paraformaldehyde for 20 min. For staining, PE-conjugated anti-human IFN-γ antibody (BD Biosciences) was added to cells for 30 min and washed with PBS containing 0.5% FBS. The stained cells were subjected to flow cytometric analysis on a FACSCalibur cytometer (BD Biosciences) and analyzed via CELLQuest software (BD Biosciences).
For apoptosis analysis, ID8 cells were collected and incubated in an annexin V-FITC/propidium iodide (PI) cell apoptosis detection kit (Sigma, USA). Briefly, cells were resuspended with 200 μL binding buffer and incubated with 5 μL annexin V (conjugated with FITC or APC) in the dark for 15 min at 37°C. Finally, the cells were stained with PI or V450 at RT for 15 min, followed by flow cytometric analysis using a FACSCalibur flow cytometer and CELLQuest software (BD Biosciences).
For apoptosis analysis, ID8 cells were collected and incubated in an annexin V-FITC/propidium iodide (PI) cell apoptosis detection kit (Sigma, USA). Briefly, cells were resuspended with 200 μL binding buffer and incubated with 5 μL annexin V (conjugated with FITC or APC) in the dark for 15 min at 37°C. Finally, the cells were stained with PI or V450 at RT for 15 min, followed by flow cytometric analysis using a FACSCalibur flow cytometer and CELLQuest software (BD Biosciences).
10
Cell Cycle Analysis by Flow Cytometry
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After being added to 75% ice-cold ethanol, 2×105 cells were incubated at 4 ℃ overnight. Next, 1 mL of DNA staining solution and 10 µL of permeabilization solution (BD, USA) were added to the samples, which were then analyzed by flow cytometry.
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