Fig. 2: Characterization of micropipette samples.
From: Image-seq: spatially resolved single-cell sequencing guided by in situ and in vivo imaging
a, Tiled, maximum intensity projection image of a 4 × 8 mm region of skull bone marrow (tiles shown as magenta squares on the schematic of the skull below the image), obtained by stitching together individual microscope fields of view. Green, β-actin-GFP-positive bone marrow cells; gray, second-harmonic generation (SHG) bone signal. White square: region of interest that is shown in the insets before and after cell isolation. Scale bars: main image, 1 mm; insets, 100 μm. Tiled calvarium bone marrow images were obtained from three independent mice. b, Average cell viability for the calvarium (MC), from a total of nine micropipette samples in n = 3 mice. Cell viability for the tibia (MT), from four micropipette samples in n = 2 mice. c, 3D visualization of a target location before and after cell aspiration. The bottom panel is obtained by rotating the top panel by 180° around the x axis. Isolated cell volume is marked by an orange outline, and the extracted cell volume (diameter ~80 µm, height 50 µm) contained in a typical micropipette sample is outlined by an orange cylinder. d, Averaged cell proportions from flow cytometry analysis of 15 calvarial micropipette (M) and 8 whole calvarial bone marrow (W) preparations in a total of n = 10 mice, and 14 tibia micropipette (M) and 5 whole tibia bone marrow (W) preparations in a total of n = 7 mice. Cells were classified based on their cell surface properties as T cells: Ter119−CD71−, CD11b−, CD3+; mature B cells: Ter119−CD71−, CD3−, CD11b−, B220+, CD19+, cKit− IgM+; pro-B cells: Ter119−CD71−, CD3−, CD11b−, B220+, CD19+, IgM−, cKit+; pre-B cells: Ter119−CD71−, CD3−, CD11b−, B220+, CD19+, IgM−, cKit−; Pre-Pro-B cells: Ter119−CD71−, CD3−, CD11b−, CD19−, B220+; macrophages: Ter119−CD71−, CD3−, CD11b+, Ly6C−, F4/80+; inflammatory monocytes: Ter119−CD71−, CD3−, CD11b+, F4/80−, Ly6CHigh; patrolling monocytes: Ter119−CD71−, CD3−, CD11b+, F4/80−, Gr1−, Ly6CLow; granulocytes: Ter119−CD71−, CD3−, CD11b+, F4/80−, Ly6CLow, Gr1+. See Extended Data Fig. 2 for the gating strategy. e, Maximum intensity projection image of a bone marrow region before and after stromal cell aspiration with the micropipette. Red, CXCL12-DsRed bone marrow cells; gray, SHG bone signal. Scale bar, 100 µm. Fifty biological replicates of stromal cell extraction were performed in 14 independent mice.
