Axiocam ic camera

HeLa cells were seeded in 6-well plates, transfected as described, and cultured until 100% confluent. Straight scratches were made across the cell layer using a 0.2 ml pipette tip. The cells were then gently washed three times with PBS to remove cellular debris and the media was replaced at 0 and 17.5 hours. Photographs of the wound region were taken using a Zeiss AxioObserver A1 microscope and AxioCam IC camera. The wound area was calculated using Image J software.

Culture samples (1 mL) were withdrawn with a syringe and transferred into 2 mL anaerobic vials, containing N₂/CO₂ atmosphere. The cells were fixed with formaldehyde 4% (in phosphate saline buffer, PBS; pH 7.2), for 15 min at room temperature. Subsequently, the samples were centrifuged at 4000 × g for 3 min and washed three times in PBS. The fixed cells were stained with 50 µg mL⁻¹ of 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 h, at 4 °C. The stained cells were washed twice in saline solution (NaCl, 0.85%) and resuspended in distilled water. Cell suspensions (5 µl) were spread on glass slides, air dried and analysed under 100 × /1.3 oil objectives by an Axio Imager M2 epifluorescence microscopy, coupled to AxioCam IC camera (Carl Zeiss Vision Inc., San Diego, USA). Simultaneous detection of three fluorophores Blue/Green/Red was carried out with the filter set 62 HE BFP + GFP + HcRed shift free (E) (Carl Zeiss Vision Inc.). Three band pass filters were applied for excitation (350–390, 460–488, 567–602) and emission (402–448, 500–557, 615–4095). Images were analysed with the software ZEN 2.3 (Carl Zeiss Microscopy GmbH, 2011). To demonstrate that the fluorescence observed was not related to the autofluorescence emitted by methanogens, in Fig. S1 we provide quality control microscopy images of non-phosphate stressed cells.

Giemsa-Wright staining was performed according to the manufacturer’s instructions (Sigma Aldrich). Briefly, 10⁵ cells in 300 μL media were spun onto a Cytospin slide and subsequently fixed with methanol for 15 min. After drying, slides were incubated with 500 µL of Wright solution for 2 min, rinsed with dH₂O, dried again, and incubated with a 1:20 Giemsa solution diluted with dH₂O for 1 h. Afterwards, the slides were thoroughly washed with dH₂O and dried. Imaging was performed using a Zeiss Axiovert200M microscope (Oberkochen, Germany), a Plan-Apochromat 63 × oil objective (Zeiss) and an AxioCam IC camera (Zeiss).