Extended Data Fig. 1: B. schlosseri cell sorting workflow.
From: Complex mammalian-like haematopoietic system found in a colonial chordate
a, Outline of cell purification process. Unsorted cells (light microscopy) are loaded into a FACS (i) and sorted, and this is followed by morphological observation (ii). Cells were labelled with diverse markers and screened using CyTOF (iii) for differential labelling. On the basis of SPADE cluster analysis (iv) markers were selected for FACS gating (v) before a final sort was performed (vi; c). b, Sorting based on FCS/SSC in the lower panel, and natural fluorescence in the upper panels. The analysis is after gating propidium iodide (PI)-negative cells (live cells). The specific excitation laser and optical filter for emission measurements were as follows (excitation laser in nm and filters stated as long pass (LP) and band pass (BP)): 488 nm for FSC and SSC, 488 nm (550LP 575/25BP)- PE, 405 nm (450/50BP)-Pacific Blue, 633 nm (660/20BP)-APC, 633 nm (690LP 710/50BP)-APC-Cy5.5. Nomenclature based on published work3,26. Experiment was performed three times. Scale bars, 20 μm. c, Sorting panels of 34 cell populations using FSC, SSC (central panel), CD49d, CD57, concanavalin-A (ConA), BHF and AP, after gating PI-negative cells (live cells). Central panel is FCS/SSC, from which additional populations were differentiated. The specific excitation laser and optical filter for emission measurements were as follows: 488 nm (505LP 530/30BP)-AP, 488 nm (755LP 780/60BP)-CD49d, 405 nm (450/50BP)-CD57, 633 nm (660/20BP)-ConA, 633 nm (755LP 780/60BP)-BHF. d, Hierarchy of sorted cell populations by main parameters of differentiation. For example, the control populations used in Fig. 2b–e (CP19, 20, 21) are all included in CP18. CP18 is derived from CP9. e, H&E staining of the end point cell populations isolated in a rectangle of original population by FSC/SSC. Live imaging was done three times; H&E was one experiment with three replicates. Colour key (c, d) for different populations identified in this study and the figures that further describe them.
