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Fig. S6
GFP expression in Tg(il1b:GFP-F) line recapitulates endogenous il1b mRNA expression in activated leukocytes
Trypsinization of 4dpf larvae was used to generate single cell suspensions for FACS sorting, and was found to activate macrophages and neutrophils, allowing us to compare endogenous il1b transcription and GFP expression triggered by a stimulus irrespective of an infection, (A-C) To test whether Tg(il1b:GFP-F) can be used to study the dynamic expression of il1b in activated leukocytes, macrophages (macro), neutrophils (neutro), and non leukocyte cells (neg) were FACS sorted from Tg(mpx:GFP; mpeg1:mCherryF) 4dpf-larvae first injected with either PBS or E. coli. Q-RT-PCR was used to measure the relative expression of mpeg1 (A) and mpx (B) in the different populations, confirming purity of sorted populations. Measurement of il1b expression (C) shows that trypinization and cell dissociation of larvae induced high level of il1b expression in macrophages and neutrophils as compared to non leukocyte cells in both PBS and E. coli injected embryos. Quantifications using ef1a as a reference gene; light grey bar represent 95% confidence interval (student T test). Non-overlapping light grey means p<0.05. (D-E) Using similar conditions to dissociate the embryos, leukocytes were cultured from Tg(il1b:GFP-F; mpeg1:mCherryF) (D) or Tg(il1b:GFP-F; Lyz:DsRed) (E). While no expression of GFP was observed in leukocytes before dissociation (in living larvae, not shown), 90% of the macrophages (red, D) and 100% of the neutrophils (red, E) expressed GFP (green) (arrowheads), showing that Tg(il1b:GFP-F) is a good reporter transgene in leukocytes. Confocal images, Scale bar= 20μm