2 ml of residual peripheral venous blood was obtained on admission from blood samples for clinical use, stored at 4 in a ethylenediamine tetraacetic acid (EDTA) tube and flow cytometry analysis was performed within 2 hours. PD-L1-PE (Cat# 560176) and CD14-APC-H7 (Cat# 560180) and their respective isotype controls recommended were purchased from BD Pharmingen (San Jose, CA, USA). 100 µl of whole blood sample was incubated with anti-CD14 and anti-PD-L1 antibodies for 20 minutes. Red blood cells were lysed. Anti-CD14 and anti-PD-L1 labeled cells and their isotypes labeled cells were obtained and run on a Gallios Flow Cytometer (Beckman Coulter, Inc.). Kaluza analysis version 1.5a (Beckman Coulter, Inc.) software was used for data analysis. Gating protocols of PD-L1 on CD14+ monocytes are shown in Figure 1 .
Kaluza analysis version 1.5a
Manufactured by Beckman Coulter
Sourced in Germany
Kaluza Analysis version 1.5a is a software application developed by Beckman Coulter to analyze flow cytometry data. It provides tools for data visualization, gating, and statistical analysis of cell populations.
Automatically generated - may contain errors
3 protocols using kaluza analysis version 1.5a
1
Analyzing PD-L1 Expression on Monocytes
2
Annexin-FITC Apoptosis Measurement
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Apoptosis was measured using an Annexin-FITC Apoptosis detection kit. The analysis was performed according to the manufacturer’s protocol. All samples were analyzed using a FACS Canto II Analytical Flow Cytometer (BDE Biosciences). The results were plotted using Kaluza Analysis version 1.5a (Beckman Coulter, Inc).
3
Quantifying Phosphorylated p70S6k by Flow Cytometry
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Flow cytometry was performed on a Navios Flow Cytometer (Beckman Coulter, Krefeld, Germany) and data were analyzed using Kaluza Analysis version 1.5a software (Beckman Coulter, Krefeld, Germany). The level of phosphorylated p70S6k was quantified using the following formula: MFIx = [(MFIp70S6K − MFIIgG2a)/MFIIgG2a].
Statistical analysis for the flow cytometry assays was performed using GraphPad Prism version V 6.01 software (GraphPad Software, La Jolla, CA, USA). A normal distribution of the measured values was not assumed. The median MFIx values were compared with the Mann–Whitney U test. Data are depicted as median with the respective interquartile range (IQR). Correlation analyses were performed using the Spearman’s test. A one-tailed p-value < 0.05 was considered as statistically significant.
Statistical analysis for the flow cytometry assays was performed using GraphPad Prism version V 6.01 software (GraphPad Software, La Jolla, CA, USA). A normal distribution of the measured values was not assumed. The median MFIx values were compared with the Mann–Whitney U test. Data are depicted as median with the respective interquartile range (IQR). Correlation analyses were performed using the Spearman’s test. A one-tailed p-value < 0.05 was considered as statistically significant.
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