Molecular probes celltracker green cmfda dye

Classical (CL) and non-classical (NC) monocytes were isolated from peripheral blood using the protocol described in Jin et al. (29 (link)) (Supplemental Figure 1). Briefly, CL (CD14++CD16–) monocytes were purified using the Human Pan-Monocyte Isolation Kit (Miltenyi) with modification of adding anti-CD16-biotin (Miltenyi) into the biotin–antibody cocktail. CD14+ selection (Miltenyi) was used subsequently to further increase purity. Purified CL monocytes were stained with Molecular Probes CellTracker Green CMFDA Dye (Life Technologies). NC (CD14^dimCD16+) monocytes were purified similarly, using CD16 microbeads (Miltenyi) during positive selection. Purity checked by flow cytometry was very high (>95%) for both CL and NC monocytes (Supplemental Figure 1).

Single-cells from each bulk monocyte subset were isolated using Fluidigm C1 Single-Cell Auto Prep System. Purified CL monocytes were stained with Molecular Probes™ CellTracker™ Green CMFDA Dye (Life Technologies), while NCL monocytes were unstained before loading to C1 Single-Cell Auto Prep Array Integrated Fluidic Circuits (IFCs). CL and NCL monocytes were then sequentially loaded onto the C1 Integrated Fluidic Circuit (IFC). CL vs. NCL monocyte lineage of individual cells was determined by direct visualization using fluorescent microscopy, and at the same time, empty wells and wells that contained more than one cell were marked to exclude from later analysis. The IFCs were then examined using fluorescent microscopy, and the captured cells were identified as CL (stained) or NCL (not stained). Wells that contained more than one cell were also noted to exclude from later analysis. We captured 470 CL and 394 NCL cells from the SLE patients in total, averaging between 50 to 60 single cells per patient across both monocyte subsets, after excluding doublets and fragments. These results represent a 60% capture site efficiency.