Mitotracker far red

At day 21 of differentiation, human iPSC-derived cardiomyocytes on 13 mm tissue-culture treated coverslips were rinsed with PBS and treated with 100 nM Mitotracker Far Red (Invitrogen P36970) for 35 min at 37°C. Coverslips were fixed in 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) at 4°C for 15 min, rinsed and stored in PBS. Cells were permeabilized with 0.5% Triton-X100 (Millipore 648466) for 10 min at room temperature, rinsed in PBS, blocked in 3% Bovine-Serum Albumin in PBS-Tween 20 0.1% for 90 min at room temperature, and rinsed in PBS. They were incubated overnight at 4 °C in 1:500 Mouse-anti-α-Actinin (Sigma A7811) and 1:300 Rabbit-anti-Pan-Cadherin (Applied Biosystems AB16505) in 3% BSA in PBS-Tween 0.1% at 4°C overnight. Coverslips were washed on a rocker at room temperature 3X for 5 min in PBS-Tween 0.1%, incubated for 1 hour at room temperature in secondary antibodies of 1:400 Donkey-anti-Mouse-488 (Invitrogen A21202) and 1:400 Donkey-anti-Rabbit-568 (Invitrogen A10042), and washed 3X for 5 min at RT on a rocker in PBS-Tween 0.1%. Cells were then stained in 1 μg/ml DAPI for 10 min at room temperature. Coverslips were mounted in Prolong Diamond (Invitrogen P36970) under a #1.5 coverslip. Imaging was done on a Nikon A1R with ×20 objectives.

B. subtilis was incubated in Lysogeny Broth (LB, Miller) and grown O/N shaking (220 rpm) at 37°C. The culture was diluted 200 times in fresh LB and allowed to grow for 2 hr at 30°C. The culture was induced with d‐Xylose (1% w/v, Sigma‐Aldrich) for 30 min before the cells were washed in PBS containing 0.5 ug/mL Mitotracker Far Red (Invitrogen). The cells were washed again in PBS and where immobilized on 1% agarose in PBS as described before (de Jong, Beilharz, Kuipers, & Veening, 2011) for microscopy.
S. aureus SH1000 carrying pLOW‐parB‐m(sf)gfp was grown in BHI broth (Oxoid) containing 5 µg/ml of erythromycin overnight. The culture was diluted 100 times in fresh medium containing 100 µM of IPTG and incubated for 2.5 hr. Cells were then immobilized on 1% agarose in PBS for microscopy.
S. pneumoniae with DnaX‐GFP (strain VL369/RR23) and DnaX‐GFP/FtsZ‐RFP (strain VL469/MK396) were grown as described before (van Raaphorst et al., 2017). S. pneumoniae strain VL451/MK359 was grown in C+Y medium recipe 2018 (Domenech et al., 2018) until an OD of 0.1 and diluted 100 times in fresh medium containing 0.1 μM of ZnCl₂. The culture was grown up to OD 0.1 again and immobilized on 1% agarose in PBS as described before for microscopy (de Jong et al., 2011).