Figure 5. Flow cytometry analysis of pigment content.

A) FACS profile of B16F2 cells with or without stable GFP expression (two left-hand panels). Effect of H89 or α-MSH on violet laser stimulated 450nm emission is shown by contour plots. B) (i) B16F2-GFP CAAX tumour cells isolated from xenograft tumours in nude mice. Contour plots show GFP (530-30nm) against Violet (450-40nm) emission. Blue line shows gate used to sort GFP expressing cells and magenta lines show the gates used to sort the top and bottom 10% pigmented cells. (ii) Pigment high and low populations were re-introduced into the tail vein of mice and the resulting lung colonies were imaged. Representative images are shown with NIRVE signal in black and GFP in green. (iii) Quantification of pigment levels assessed on a cell by cell basis in colonies generated from pigment high and low populations. Relative frequency of pigment levels is shown (data from 12 mice in three independent experiments). C) (i) B16F2 Brn2::GFP/mRFP-CAAX tumour cells isolated from xenograft tumours in nude mice. Contour plots show Red (620-40nm) against GFP (530-30nm) emission. Blue line shows gate used to sort mRFP expressing cells and green lines show the gates used to sort the top and bottom 10% Brn2::GFP cells. (ii) Brn2::GFP high and low populations were re-introduced into the tail vein of mice and the resulting lung colonies were imaged. Two representative images are shown with mRFP-CAAX signal in red and Brn2::GFP in green. * highlights three cells that have converted back to high levels of Brn2::GFP expression. (iii) Quantification of Brn2::GFP levels assessed on a cell by cell basis in colonies generated from Brn2::GFP high and low populations. Relative frequency of Brn2::GFP levels is shown (data from 10 mice in three independent experiments). *indicates p<0.01 Mann-Whitney test.