Peripheral blood was collected by cardiac puncture into EDTA tubes. BM cells were isolated by centrifuging femurs (13,000g for 15 minutes at 4°C). Liver tissues were homogenized gently using a 3 mL syringe plunger. RBCs were lysed (ACK Lysing Buffer, Thermo Fisher Scientific). Any clumps of cells were removed by filtration using 70 μm strainers (Thermo Fisher Scientific). Cells were washed and kept in PBS supplemented with 2% FBS and were stained with the following fluorochrome-conjugated antibodies: CD45 (Alexa Fluor 700, BioLegend, 103128, 1:300), CD11b (Brilliant Violet 421, BioLegend, 101235, 1:500), Ly6G (APC, BioLegend, 127614, 1:300; FITC, BioLegend, 127605, 1:500), CD11c (Alexa Fluor 488, BioLegend, 117313, 1:100; PE-Cyanine7, BioLegend, 117318, 1:100), TLR2 (PE, BioLegend, 148604, 1:200), TLR4 (PE-Cyanine7, BioLegend, 117610, 1:200), TLR1 (PE, Thermo Fisher Scientific, 12-9011-80, 1:100), and TLR5 (APC, BioLegend, 148105, 1:100) at 4°C and washed twice. Intracellular staining for HO-1 was performed by permeabilizing the cells using the BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) and staining the cells with antibody against HO-1 (ab52947, 1:200), followed by donkey anti-rabbit secondary antibody (BioLegend, 406414, 1:500).
Pe cyanine7
Manufactured by BioLegend
Sourced in United States
PE/Cyanine7 is a fluorescent dye conjugate. It is used for multicolor flow cytometry and cell imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Apc rat igg2a, by BioLegend (3 mentions)
Pe cyanine7 rat igg2b, by BioLegend (3 mentions)
Pe rat igg2b, by BioLegend (3 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (3 mentions)
Fitc rat igg2a, by BioLegend (3 mentions)
Anti cd16 cd32 antibody, by BioLegend (3 mentions)
Efluor 780, by Thermo Fisher Scientific (3 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (3 mentions)
Brilliant violet 421, by BioLegend (2 mentions)
Alexa fluor 700, by BioLegend (2 mentions)
Percp cyanine5, by BioLegend (2 mentions)
Allophycocyanin, by BioLegend (2 mentions)
Peridinin chlorophyll protein cyanine 5, by BioLegend (2 mentions)
Facsverse, by BD (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc, by BioLegend (1 mentions)
Trypletm, by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Percp, by BioLegend (1 mentions)
Apc cyanine7, by BioLegend (1 mentions)
12 protocols using pe cyanine7
1
Multiparametric Analysis of Innate Immune Cells
2
Annexin V Apoptosis Assay in Cancer Spheroids
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Cancer spheroids and T cells were co-cultured for 4 d until all cells were collected from the agarose casts.15 (link) After single-cell dissociation with TrypLETM (# 12604013, ThermoFisher), Annexin V staining was performed. T cells were separated from the cancer cells by gating on CD3+ (# 100209, BioLegend) population. Annexin V FITC (# 640945, BioLegend) or PE/Cyanine 7 (# 640951, BioLegend) was used according to the manufacturer’s recommendation. All tests were performed in duplicates or triplicates.
3
Multiparameter Flow Cytometry of Immune Cells
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Cells were stained with the following fluorochrome-conjugated antibodies: TLR2 (APC, BioLegend, 309719, 1:100; FITC, BioLegend, 309705, 1:100), TLR4 (PE, BioLegend, 312805, 1:100), CD15 (FITC, BioLegend, 301903, 1:100), CD16 (Alexa Fluor 700, BioLegend, 302025, 1:100), CD11b (Brilliant Violet 421, BioLegend, 101235, 1:100), CD66b (PE-Cyanine7, BioLegend, 305115, 1:100). The stained cells were run on CytoFLEX Flow Cytometer (Beckman Coulter). Flow cytometry data were analyzed by FlowJo software. Flow cytometry gating strategy is shown in Supplemental Figure 5 .
4
Multiparametric Flow Cytometry Analysis of T Cell Subsets
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Tumors were obtained to prepare single-cell suspension and then incubated with red blood cells lysis buffer. Cell density was adjusted to 1×106 per milliliter. Cells were incubated with anti-CD16/32 antibody to block Fc receptors. After incubating with antibody targeted cell surface antigen including anti-45, anti-CD3, anti-CD8, anti-CD4, and anti-CD25 antibodies, cells were fixed with 1× Foxp3 Fix/Perm buffer and next incubated with 1× Foxp3 Perm buffer for the detection of intracellular antigen Foxp3. Finally, cells were incubated with anti-Foxp3 antibody in the dark at room temperature and analyzed with CytoFlex S (Beckman). The antibodies used were as follows: anti-CD45 antibody (MultiSciences, Violetflour 450, cat.70-AM04512-100; BioLegend, APC/Cyanine7, cat.103116), anti-CD3ϵ antibody (BioLegend, PE/Cyanine7, cat.100320; BioLegend, PerCP/Cyanine5.5, cat.100328), anti-CD4 antibody (BioLegend, APC, cat.100412), anti-CD8a antibody (BioLegend, PerCP, cat.100732; BioLegend, FITC, cat.100706), and anti-Foxp3 antibody (BioLegend, PE, cat.320008).
5
Multi-Organ Immune Cell Analysis
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Single cell suspensions (1 × 106 cells) obtained from different organs were pre-incubated with anti–CD16/CD32 (93) to block FcγRII/III receptors and stained with the desired antibodies (Abs) on ice for 30 minutes and analyzed on FACSVerse (BD Bioscience, San Jose, CA, USA). The following anti-mouse Abs conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein-cyanine 5.5, allophycocyanin or PE-cyanine7 (all from Biolegend, San Diego, CA, USA) were used for staining: anti-Gr-1 (RB6-8C5) and anti-CD11b (M1/70), anti-CD4 (GK1.5/RM4-5), anti-CD8α (53–6.7), anti-TCRβ (H57-597), anti-NK1.1 (PK136). Dead cells were excluded by forward light scatter or forward light scatter plus propidium iodide. All the data were acquired and presented on log scale. Data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).
6
Skin Wound Immune Cell Analysis
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Single cells digested from skin wounds were pre-incubated with purified anti-CD16/CD32 antibody (101301, BioLegend) (1.0 μg per 106 cells in 100 μl volume) for 5 to 10 min to block Fc receptors. The cell suspensions were then co-incubated with fixable viability dye (eFluor™ 780, 65-0865-14, eBioscience) and antibodies against surface markers CD45 (PE/Cyanine7, 147703, BioLegend), CD3 (PE, 100205, BioLegend), and F4/80 (FITC, 123107, BioLegend) at 1:400 dilution for 30 min at 4 °C in the dark (100 μl per antibody per sample). After fixation and permeabilization, cells were incubated with antibodies against intracellular marker CD68 (APC, 137007, Biolegend) at 1:400 dilution for 20 min at 4 °C in the dark (100 μl per antibody per sample). Isotype controls of CD45 (PE/Cyanine7 Rat IgG2b, κ, 400617, Biolegend), CD3 (PE Rat IgG2b, κ, 400607, Biolegend), F4/80 (FITC Rat IgG2a, κ, 400505, BioLegend) and CD68 (APC Rat IgG2a, κ, 400511, Biolegend) were used in same concentration. Flow cytometry analysis was performed using Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo (v10.8.1). The experiments were performed three times independently (n = 3).
7
Isolation and Characterization of Lung Immune Cells
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We prepared single-cell suspensions from collagenase-treated lungs, which we strained through 70 mm mesh and centrifuged through 44% Percoll (MP Biomedical, https://www.mpbio.com ) in Gibco RPMI 1640 medium (Thermo Fischer Scientific, https://www.thermofisher.com ), and then layered onto 67% Percoll in 1× PBS. We used TruStain FcX (Biolegend, https://www.biolegend.com ) anti-mouse CD16/32 antibody to block Fc receptor cells and performed flow cytometry by using the LSR II system (BD Biosciences, https://www.bdbiosciences.com ). We then stained surface markers with the antibodies used to sort neutrophils, T cells, B cells, and natural killer (NK) cells. We used the following Biolegend products from Thermo Fischer Scientific: for neutrophils, CD11b (CD11b Antibody, PE-Cyanine 7), CD115 (CD115 Antibody, APC), lymphocyte antigen complex 6 locus G (Lys6G Antibody, AF488); for T cells, CD45 (CD45 Antibody, Alexa Fluor-700), CD3 (CD3 Antibody-APC); for B cells, B220 (B220 Antibody-PE-Cy7), and NK cells, NK1.1 (NK1.1 Antibody-PE) (Appendix Figure 1). We analyzed data by using FACS Diva version 8.0.1 (BD Biosciences) and determined percentage viability by using Zombie Aqua (Biolegend) live-dead dye.
8
T-Cell Immunophenotyping and Cytokine Analysis
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Cells were washed with FACS buffer (PBS buffer supplemented with 1% FCS and 1 mM EDTA) by centrifugation and stained with the respective antibodies. Reactions were incubated for 30 min at 4 °C. Following two washes with FACS buffer, samples were resuspended in 200 μl of FACS buffer and data was acquired using a Fortessa analyser (BD Bioscience). All data were processed with FlowJo (v9.9, TreeStar). Antibodies used in this study: anti-human CD4, APC (BioLegend; Clone: OKT4, Catalog No: 317416, Dilution: 1:100), anti-human CD45RA, Brilliant Violet 785 (BioLegend; Clone: HI100, Catalog No: 304140, Dilution: 1:100), anti-human CD45RO, PE-Cyanine7 (BioLegend; Clone: UCHL1, Catalog No: 304229, Dilution: 1:100), anti-human CD197 (CCR7), (BD Bioscience; Clone: 150503, Catalog No: 561271, Dilution: 1:100), anti-human IFN gamma, PE-Cyanine7 (eBioscience; Clone: 4s.B3, Catalog No: 25-7319-82, Dilution: 1:50), anti-human IL-9, PE (BD bioscience; Clone: MH9A3, Catalog No: 560814, Dilution: 1:50).
9
Skin Wound Immune Cell Analysis
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Single cells digested from skin wounds were pre-incubated with purified anti-CD16/CD32 antibody (101301, BioLegend) (1.0 μg per 106 cells in 100 μl volume) for 5 to 10 min to block Fc receptors. The cell suspensions were then co-incubated with fixable viability dye (eFluor™ 780, 65-0865-14, eBioscience) and antibodies against surface markers CD45 (PE/Cyanine7, 147703, BioLegend), CD3 (PE, 100205, BioLegend), and F4/80 (FITC, 123107, BioLegend) at 1:400 dilution for 30 min at 4 °C in the dark (100 μl per antibody per sample). After fixation and permeabilization, cells were incubated with antibodies against intracellular marker CD68 (APC, 137007, Biolegend) at 1:400 dilution for 20 min at 4 °C in the dark (100 μl per antibody per sample). Isotype controls of CD45 (PE/Cyanine7 Rat IgG2b, κ, 400617, Biolegend), CD3 (PE Rat IgG2b, κ, 400607, Biolegend), F4/80 (FITC Rat IgG2a, κ, 400505, BioLegend) and CD68 (APC Rat IgG2a, κ, 400511, Biolegend) were used in same concentration. Flow cytometry analysis was performed using Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo (v10.8.1). The experiments were performed three times independently (n = 3).
10
Analyzing MDSC Phenotype and Activation
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MDSCs were induced in vitro with or without SFN treatment (10 µM). After 72 hours, the cells were harvested and labeled for CD11b (PerCP/Cyanine5.5, Cat.101227 BioLegend, USA), Gr-1 (APC, Cat.108412, BioLegend, USA), cleaved caspase-3 (PE, Cat.9661, Cell Signaling Technology, USA), PD-L1 (PE/Cyanine7, Cat.124314 BioLegend, USA), and IL-12RB2 (FITC, Cat.ab96097, Abcam, USA). Imaging flow cytometry was used to measure protein levels.
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