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cmi12085-sup-0001-si.TIF304K

Fig. S1. Synthesis of fluorescent protein genes adapted for Gram-positive (gp) bacteria. The amino acid sequence of fluorescent proteins is depicted with the nucleotide sequence of the corresponding genes. The gfpmut2 gene was used as a template to introduce nucleotide exchanges (red), which lead to the intended amino acid substitutions.

cmi12085-sup-0002-si.TIF1241K

Fig. S2. Intravital 2-P microscopy of a low-dose S. aureus skin infection. A total of 103 cfu Newman-sar-VEN were injected into the dermis of a C57BL/6 mouse 30 min prior to intravital imaging of the flank skin using 2-P microscopy. The bacterial cells (yellow) are visible as being embedded in the dermal extracellular matrix (blue). Scale bar = 10 μm.

cmi12085-sup-0003-si.TIF1901K

Fig. S3. Intravital 2-P microscopy 6 h after infection. A total of 104 cfu Newman-sar-CER were injected intradermally into the skin of a LysM-EGFP mouse 6 h prior to imaging a flank skin flap by intravital 2-P microscopy. PMN (green) and S. aureus (blue) are detectable in the dermis (left panel). Time-coded cell tracks (middle panel, total duration 20 min) and displacement vectors (right panel) indicate a movement of PMN towards the focus of infection. Scale bar = 100 μm.

cmi12085-sup-0004-si.TIF37K

Fig. S4. Bacterial loads after PTx treatment. C57BL/6 mice received or not 250 μg kg−1 PTx i.p. 1 day prior to intradermal flank skin infection with 107 cfu Newman. Bacterial burdens were quantified by plating serial dilutions of the homogenized skin lesions on TSB agar plates 5 days after infection. n = 5 per group; error bars = SD; **P < 0.01 (t-test).

cmi12085-sup-0005-si.mov1034K

Movie S1. Growth of fluorescent S. aureus reporter strains on agar plates. RN6734-sar-GFP (green) and RN6734-agr-CER (blue) were seeded on TSB agar plates and fluorescence and transmitted light of micro-colonies were recorded for 13 h by confocal microscopy during incubation at 37°C. Time is displayed as d:h:min:s:ms.

cmi12085-sup-0006-si.mov160K

Movie S2. GFPlo cells exhibit a stationary behaviour in the skin of uninfected LysM-EGFP mice. The dermis of an uninfected LysM-EGFP mouse was examined by intravital 2-P microscopy. The time-lapse movie depicts the movement of GFPlo cells (green) in the dermal matrix (blue). Time is displayed as h:min:s. Scale bar = 20 μm.

cmi12085-sup-0007-si.mov454K

Movie S3. Neutrophils move towards the focus of infection. A LysM-EGFP mouse was intradermally infected with 104 cfu Newman-sar-CER (blue). The time-lapse movie was acquired 2 h after infection using 2-P microscopy. PMN (green) are moving through the dermal tissue towards the infection site. Time is displayed as h:min:s. Scale bar = 50 μm.

cmi12085-sup-0008-si.mov533K

Movie S4. A bacterial cluster is not taken up by PMN. A LysM-EGFP mouse was infected with 106 cfu Newman-sar-CER and the vicinity of the infection focus was observed by 2-P microscopy after 6 h. A high magnification time-lapse movie shows S. aureus cells and clusters (blue), which are probed by PMN (green). Time is displayed as h:min:s. Scale bar = 10 μm.

cmi12085-sup-0009-si.mov2342K

Movie S5. PMN accumulate at site of S. aureus infection. A LysM-EGFP mouse was infected with 104 cfu Newman-sar-CER 6 h prior to intravital imaging using 2-P microscopy. PMN (green) stream towards and accumulate at the site of the S. aureus deposit (blue). Time is displayed as h:min:s. Scale bar = 50 μm.

cmi12085-sup-0010-si.mov1433K

Movie S6. Development of two zones during abscess formation. A LysM-EGFP mouse was infected with 106 cfu Newman-sar-CER 1 day before intravital imaging using 2-P microscopy. PMN (green) have accumulated and exhibit little movement, whereas they migrate in the periphery. S. aureus (blue) can be observed within and outside the developing abscess. Time is displayed as h:min:s. Scale bar = 100 μm.

cmi12085-sup-0011-si.mov343K

Movie S7. PTx inhibits emigration of PMN in response to S. aureus infection. A LysM-EGFP mouse was treated with 250 μg kg−1 PTx 1 day prior to infection with 106 cfu Newman-sar-CER. Six hours later, a blood vessel in the vicinity of the infection focus was analysed by 2-P microscopy. The blood vessel was made visible by injection of Qdots (red) immediately before imaging. PMN (bright green) can be observed in the bloodstream, while GFPlo cells (dim green) and S. aureus (blue) are located in the dermal matrix. Time is displayed as h:min:s. Scale bar = 20 μm.

cmi12085-sup-0012-si.mov334K

Movie S8. Transfer of labelled bone-marrow PMN. Bone-marrow PMN were purified from a C57BL/6 mouse, labelled in vitro with 5 μM SNARF-1 and 2 × 106 cells were transferred i.v. into a LysM-EGFP mouse 15 min prior to infection with Newman-sar-CER. Four hours later the vicinity of the infection focus in the dermis was analysed by intravital 2-P microscopy. Transferred PMN (red) can be observed among the endogenous PMN (green). Location of the infection focus is indicated. Time is displayed as h:min:s. Scale bar = 50 μm.

cmi12085-sup-0013-si.mov314K

Movie S9. Transfer of WT and IL-1R−/− PMN. Bone-marrow PMN were isolated from C57BL/6 WT and IL-1R−/− mice and labelled in vitro with 5 μM SNARF-1 or CFSE. A total of 4 × 106 cells (mixed at a 1:1 ratio) were intravenously transferred into a C57BL/6 mouse 15 min before infection with 104 cfu Newman-sar-CER. Time-lapse movie was acquired 6 h after infection. WT PMN (red) and IL-1R−/− PMN (green) have migrated to the site of S. aureus infection (blue). Time is displayed as h:min:s. Scale bar = 50 μm.

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