CD163 Antibody, anti-human, PE-Vio® 770, REAfinity™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A total of 0.1 ml of peripheral blood was incubated with 10 μl of antibody for 15 min at room temperature, then erythrocytes were lysed and leucocytes post-fixed. Afterwards, the flow cytometry analysis was performed.	Flow cytometric analysis was performed using a Navios Flow Cytometer and the Kaluza analysis software (Beckman Coulter, Milan, Italy), evaluating a total of 5 × 106 cells and detecting more than 30 events in the smallest subset investigated, according to consensus guidelines on the minimal residual disease

Protocol tips
A total of 0.1 ml of peripheral blood was incubated with 10 μl of antibody for 15 min at room temperature, then erythrocytes were lysed and leucocytes post-fixed. Afterwards, the flow cytometry analysis was performed.
Downstream tips
Flow cytometric analysis was performed using a Navios Flow Cytometer and the Kaluza analysis software (Beckman Coulter, Milan, Italy), evaluating a total of 5 × 106 cells and detecting more than 30 events in the smallest subset investigated, according to consensus guidelines on the minimal residual disease

Publication protocol

"After enrolment, peripheral blood was collected in a lithium-heparin single tube from each SSc patient.

To identify monocyte/macrophage lineage surface markers CD14-APC-Vio770 and CD45-VioGreen antibodies were used. The characterization of M2 phenotype was performed using CD204-PE, CD163-PE-Vio770 and CD206-PeerCP-Vio700, whereas the M1 phenotype was investigated using CD80-APC, CD86-VioBlue, TLR2-PE-Vio615 and TLR4-VioBright-FITC antibodies. CD66b-FITC was used to identify and exclude granulocytes (Miltenyi Biothech, Bergisch Gladbach, Germany).

A total of 0.1 ml of peripheral blood was incubated with 10 μl of antibody for 15 min at room temperature, then erythrocytes were lysed and leucocytes post-fixed. Afterwards, the flow cytometry analysis was performed.

Three initial gating strategies were implemented to investigate circulating monocyte/macrophage phenotype over total leucocyte population and included in the Additional file 1. The first initial gating strategy evaluated the CD14+ cells over total leucocyte population. In this CD14+ cell population, circulating monocytes/macrophages showing an M2 phenotype were characterized based on the expression of CD204, CD163 and CD206. Therefore, a second initial gating strategy evaluated the CD204+ cells in the leucocyte population, excluding lymphocytes, CD66b+ granulocytes, doublets and cellular debris. In the CD204+ population, circulating cells co-expressing CD163 and CD206 were detected to characterize monocytes/macrophages showing an M2 phenotype. Cells positive for M2 phenotype markers (CD204, CD163, CD206) and M1 phenotype markers (TLR4, CD80 and CD86) were investigated to identify the presence of cells with a mixed M1/M2 phenotype, as recently reported [22]. Although lymphocytes and neutrophils are excluded in the initial gating strategy starting from CD204 + cells, no specific dendritic cell markers were investigated to discriminate these cells and then they might be probably present in a limited percentage in the M1/M2 mixed population.

Finally, a third initial gating strategy was made up to detect monocytes/macrophages showing prominently M1 surface markers CD80, CD86, TLR2 and TLR4 [27].

Flow cytometric analysis was performed using a Navios Flow Cytometer and the Kaluza analysis software (Beckman Coulter, Milan, Italy), evaluating a total of 5 × 106 cells and detecting more than 30 events in the smallest subset investigated, according to consensus guidelines on the minimal residual disease [28]."

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