Figure 4.

Sorting and isolation of single cells from two coelomocyte sub-populations and from sperm for WGA. Small phagocytes that express SpTrf on their surface and red spherule cells were sorted from animals 1–3 pre-challenge on day 0 as well as on days 1 and 2 post-challenge with Vibrio diazotrophicus. Sperm cells were collected directly from the gonopores of the same sea urchins. (A) Coelomocytes were gated from debris based on forward scatter (FSC) and side scatter (SSC). (B) Live cells were gated for propidium iodide (PI) exclusion and for surface staining of anti-SpTrf and secondary antibody conjugated with Alexa Fluor 488 (AF-488). (C) Live cells incubated with the secondary antibody alone did not show AF-488 staining. (D) Red spherule cells were gated based on their natural far-red auto-fluorescence (allophycocyanin channel; APC). Single cells with high auto-fluorescence were sorted and observed post-sorting by light microscopy (inset). (E) Live cells with high surface SpTrf protein levels were gated based on the AF-488 fluorescence of the secondary antibody. Single cells were sorted and observed post-sorting by fluorescence microscopy (inset). (F) No cells were recorded in the same gate as in (E) for the sample incubated with the secondary antibody only. This demonstrated that all cells within the gate in (E) had SpTrf on the surface and were likely small phagocytes. (G) Sperm cells were obtained by electric shock stimulation (16–20 mA), diluted to 1×10⁵ cells/ml, stained with Hoechst 33342 and serially diluted to 0.25 cells/μl. (H) Single cells of each type were isolated either by FACs or manually into 4 μl of 3.3X PBS in a 384-well plate, observed under epifluorescent microscope, and subjected to WGA using the multiple displacement amplification with the REPLI-g single-cell kit (Qiagen). The scale bars in the insert figures in (D,E) are 10 μm.