PE Mouse Anti-Human CD163

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Cells were then detached with accutase (Sigma-Aldrich), washed in PBS, and incubated with 100 µL of blocking buffer [PBS containing 10% human AB serum (Sigma-Aldrich), 2% FCS (Lonza), and 0.02% NaN3 (Sigma-Aldrich)]. Cells were then labeled in brilliant stain buffer (BD Bioscience) with a combination of fluorescently conjugated monoclonal antibodies	Flow cytometry analysis was performed on a BD LSRFortessa instrument using FACSDiva software (BD Biosciences), with 10,000 events acquired for each sample.

Protocol tips
Cells were then detached with accutase (Sigma-Aldrich), washed in PBS, and incubated with 100 µL of blocking buffer [PBS containing 10% human AB serum (Sigma-Aldrich), 2% FCS (Lonza), and 0.02% NaN3 (Sigma-Aldrich)]. Cells were then labeled in brilliant stain buffer (BD Bioscience) with a combination of fluorescently conjugated monoclonal antibodies
Downstream tips
Flow cytometry analysis was performed on a BD LSRFortessa instrument using FACSDiva software (BD Biosciences), with 10,000 events acquired for each sample.

Publication protocol

PB monocytes (106 cells/well) were plated in six-well plates and incubated for 72 h with the polarizing stimuli at a final concentration of 5% FBS. They were then detached with accutase (Sigma-Aldrich), washed in PBS, and incubated with 100 µL of blocking buffer [PBS containing 10% human AB serum (Sigma-Aldrich), 2% FCS (Lonza), and 0.02% NaN3 (Sigma-Aldrich)]. Cells were then labeled in brilliant stain buffer (BD Bioscience) with a combination of fluorescently conjugated monoclonal antibodies against HLADR, CD80, CD23, CD206, and CD163 (BD Biosciences). Flow cytometry analysis was performed on a BD LSRFortessa instrument using FACSDiva software (BD Biosciences), with 10,000 events acquired for each sample. Integrated median fluorescence intensity (iMFI) was computed by multiplying the relative frequency (percentage of positive) of cells expressing each marker by the median fluorescence intensity (MFI) of the cell population.

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