Quantification of infection was undertaken by flow cytometry exactly as previously described22 (link), except that analysis was undertaken on a Cyan ADP 9-color flow cytometer (Beckman Coulter, Brea, CA) and analysis was performed using Kaluza software (Beckman Coulter, Brea, CA). All experiments were undertaken independently in duplicate. Infected cells were gated as M2.
Cyan adp 9 color flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Cyan ADP 9-color flow cytometer is a laboratory instrument designed for the analysis of cells and particles. It is capable of detecting and measuring up to 9 different fluorescent markers simultaneously on individual cells or particles passing through a laser beam.
Automatically generated - may contain errors
Lab products found in correlation
Kaluza software, by Beckman Coulter (2 mentions)
Flowcytomix pro 3, by Thermo Fisher Scientific (2 mentions)
Cxcl10, by BD (2 mentions)
Ifn γ, by BioLegend (1 mentions)
Tnf α, by BD (1 mentions)
Tcs sp5, by Leica camera (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Monensin, by Merck Group (1 mentions)
Anti ifnγ percp cy5, by BioLegend (1 mentions)
Anti tnf α fitc, by BioLegend (1 mentions)
Anti il2 pe cy7, by BioLegend (1 mentions)
Anti il 10 apc, by BioLegend (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
5 protocols using cyan adp 9 color flow cytometer
1
Quantification of Viral Infection by Flow
2
Cytokine and Chemokine Profiling of Cell Supernatants
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Supernatants from cultured cells were monitored using the human Th1/Th2/Th9/Th17/Th22 13-plex RTU FlowCytomix Kit (eBiosciences), and human chemokine 6-plex kit FlowCytomix (eBiosciences) according to the manufacturer’s instructions and acquired on a Cyan ADP 9-color flow cytometer (Beckman Coulter). Analyses were performed by FlowCytomix Pro 3.0 Software (eBiosciences). Some measurements were performed by ELISA with IFNγ (BioLegend), IL-9 (BioLegend), TNFα (BD Biosciences), CCL2 (BD Biosciences), CCL3 (R&D Systems), CCL4 (R&D Systems), CCL5 (R&D Systems), and CXCL10 (BD Biosciences) kits in accordance with the manufacturer’s recommendations.
3
Cellular Internalization of Targeted Nanoparticles
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B16 cells were routinely cultured in RMPI 1640 medium with 10% FBS in a humidified incubator with 5% CO2 at 37 °C. After 24 h, cells were incubated with UN/MC540-DOX and FA-UN/MC540-DOX (10 μg/mL) at 37 °C for different time intervals. Cells were rinsed with PBS (pH 7.2) solution softly and detached by 0.2% trypsin. And removed typsin by centrifugation at 1000 rpm for 5 min. Cells were then harvested and re-suspended in 100 mL PBS and examined by flow cytometry using a CyAn ADP 9 color flow cytometer (Beckman Coulter). Using the same method, B16 cells were grown in a culture dish at a density of 1 × 106 cells/dish and incubated at 37 °C for 24 h. Cells in dishes were incubated with culture containing 200 μl of the above two kinds of nanoparticles. Then removed medium and washed cells with PBS (pH 7.2). The cell nucleus and cell membrane were stained with Hoechst and CellMask™ Deep Red Plasma membrane Stain, respectively. FA-UN/MC540-DOX was detected at 980 nm and corresponding fluorescent images at 540 nm was taken by CLSM (TCS SP5, Leica).
4
Profiling Tumor-Derived Cytokine Landscape
5
Comprehensive T Cell Cytokine Profiling
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Cryopreserved PBMCs from each participant at all time points were investigated simultaneously. After overnight rest of thawed PBMC, the cells were cultured in the presence of anti-CD107a-PE in complete RPMI containing 2 μg/mL of recombinant HBsAg (MyBioSource, USA) and 5 μg/mL of PHA (Sigma-Aldrich, USA) as the positive stimulation control or complete media alone as the unstimulated control at 37°C in a 5% CO2 incubator for 16–18 hr.
Cytokine-producing T cells analysis was performed as described previously [34 (link)]. Briefly, the overnight culture was further incubated with 5 μg/mL of brefeldin A and 1 μM of monensin (Sigma-Aldrich, USA) for 4 hr. Then, the cells were stained with anti-CD8-PE Alexa Fluor 610 (Life Technologies, USA), and anti-CD4-APC/Cy7 and anti-CD45RO-Pacific Blue (BioLegend, USA). After fixation and permeabilization, intracellular staining was performed by incubating the cells with anti-CD3-Krome Orange (Beckman Coulter, USA), and with anti-TNF-α-FITC, anti-IFN-γ-PerCP/Cy5.5, anti-IL-2-PE/Cy7, and anti-IL-10-APC (BioLegend, USA). At least 100,000 lymphocytes were collected for each sample by using Cyan ADP 9-color flow cytometer (Beckman Coulter, USA). Flow cytometric analysis of cytokine-producing or degranulation maker CD107a-expressing T cells was performed by using Kaluza software (Beckman Coulter, USA).
Cytokine-producing T cells analysis was performed as described previously [34 (link)]. Briefly, the overnight culture was further incubated with 5 μg/mL of brefeldin A and 1 μM of monensin (Sigma-Aldrich, USA) for 4 hr. Then, the cells were stained with anti-CD8-PE Alexa Fluor 610 (Life Technologies, USA), and anti-CD4-APC/Cy7 and anti-CD45RO-Pacific Blue (BioLegend, USA). After fixation and permeabilization, intracellular staining was performed by incubating the cells with anti-CD3-Krome Orange (Beckman Coulter, USA), and with anti-TNF-α-FITC, anti-IFN-γ-PerCP/Cy5.5, anti-IL-2-PE/Cy7, and anti-IL-10-APC (BioLegend, USA). At least 100,000 lymphocytes were collected for each sample by using Cyan ADP 9-color flow cytometer (Beckman Coulter, USA). Flow cytometric analysis of cytokine-producing or degranulation maker CD107a-expressing T cells was performed by using Kaluza software (Beckman Coulter, USA).
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