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Tested different loading dyes enabling to follow cell viability by fluorescence microscopy.
The ON culture of M15[pRep4-pQE30::KR] was split in two different Erlenmeyers and incubated at 37°C, 300rpm. One of them was kept in the dark while the other was illuminated (I = ) for 2 hours to induce cell death. For both samples, 200 µL were pipetted, centrifuged, and incubated 15 min (37°C, 200 rpm) with 200µL of one of the following loading dyes : 5-100µM Diaminofluorescein, 500-5nM MitoTracker Green FM (M7514, Invitrogen, Carlsbad, NM, USA), XX SYBR Safe DNA gel stain (S33102, Invitrogen). Cells were subsequently washed in 200 µL PBS, mounted on a microscope slide and observed with the epifluorescence microscope available in our lab (FITC filter cube).
Performed a new kinetics on M15[pRep4-pQE30::KR] cells, in M9 medium.
M15[pRep4-pQE30::KR] cells were cultured ON (37°C, 200 rpm) in 10 mL LB medium, supplemented with Kanamycin and Ampicillin. In the morning, cells were diluted 100x in fresh LB medium (with antibiotics). At OD610 = 1.5, cells were diluted 100x in 75 mL M9 medium, supplemented with 50 µg/mL Kanamycin, 200 µg/mL Ampicillin and 0.05 M IPTG. The solution was subsequently split in 3 Erlenmeyers, covered with aluminum fold, and incubated at 37°C, under light illumination. 195 min after IPTG induction, one of the Erlemeyer was uncovered, and second one followed the same fate 110 min later. OD610 and fluorescence (540/630 nm) measurements and cell plating were performed every 45-70 min. .
