Blood cells (80 μL) from COV, PASC and HD were vortexed for 5 min and incubated for 15 min in the dark at room temperature with 1.5 mL of VersaLyse Lysing Solution (Beckman Coulter, BC) to lyse red blood cells and select a total leukocyte population. Antibodies of interest were incubated for 15 min in the dark on ice. 10 μL of IOTest myeloid activation antibody cocktail composed of anti-CD169-phycoerythrin (PE) (clone 7–239), anti-CD64-Pacific Blue (PB) and HLA-DR (APC) (clone 22) (Beckman Coulter) was used. The stained cells were analysed via CytoFLEX (Beckman Coulter) and CytExpert 2.3 software (BC). CD169 expression was represented as the ratio of CD169 MFI in HLA-DR+ monocytes versus HLA-DR+ lymphocytes (RMFI) as described in a previous study (Minutolo et al., 2021 (link)). Furthermore, the percentage of positive monocytes for the HLA-DR and CD169 markers was also analyzed as reported in the gating strategy described in the Supplementary Material 1 .
Anti cd169 phycoerythrin pe clone 7 239
Manufactured by Beckman Coulter
Anti-CD169-phycoerythrin (PE) (clone 7-239) is a monoclonal antibody conjugated with the fluorescent dye phycoerythrin (PE). It is designed for the detection and analysis of CD169-positive cells in flow cytometry applications.
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2 protocols using anti cd169 phycoerythrin pe clone 7 239
1
Quantifying Immune Cell Activation in COVID-19
2
Immune Profiling of COVID-19 Patients
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EDTA blood samples (10 μL) were simultaneously lysed with 500 μL of Versalyse lysing solution (Beckman Coulter) and stained with CD64-CD169/infection dried custom mixture composed of anti-CD169-phycoerythrin (PE) (clone 7-239), anti-CD64-Pacific Blue (PB), and HLA-DR (APC) (clone 22) (Beckman Coulter).
The DuraClone IM T-cell subset tube and B-cell subset tube from Beckman Coulter were used to analyze differentiation and exhaustion markers. Stained cells were then washed with Dulbecco’s phosphate-buffered saline (PBS, PAN-Biotech). The stained cells were examined using a CytoFLEX (Beckman Coulter), and the data were analyzed using the CytExpert 2.2 software (Beckman Coulter). The gating strategy is reported inFigure S2 and described in previous work [12 (link),15 (link)]. In monocytes, the expression of mCD169 MFI was very high in COVID-19 patients compared to healthy donors (p = 0.010), while in lymphocytes, no significant difference was detected. Interestingly, the ratio of the CD169 MFI between monocytes and lymphocytes (RMFI) was even more significant (p < 0.001) than the MFI in monocytes alone (Figure S1A ); for this reason, all the results are expressed as the RMFI. The analysis of CD169 MFI in monocytes and lymphocytes were reported in Supplementary Figure S1 and in Supplementary Table S1 .
The DuraClone IM T-cell subset tube and B-cell subset tube from Beckman Coulter were used to analyze differentiation and exhaustion markers. Stained cells were then washed with Dulbecco’s phosphate-buffered saline (PBS, PAN-Biotech). The stained cells were examined using a CytoFLEX (Beckman Coulter), and the data were analyzed using the CytExpert 2.2 software (Beckman Coulter). The gating strategy is reported in
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