Anaerotest

H9C2 rat cardiac myoblasts were purchased from ATCC (Cat.No. CRL-1446) and cultured in Dulbecco’s modified eagle medium (DMEM; PAN) supplemented with 10 % fetal calf serum (FCS; PAN), 1 % non-essential amino acids, 1 % MEM vitamins, 1 % penicillin/streptomycin at 37 °C in humidified air containing 5 % CO₂. To mimic ischemia, cell were grown to 70 % confluency, washed three times with PBS and incubated in DMEM serum free supplied with SAR1 or DMSO for 24 h in a special incubation bag containing Anaerocult^® A mini (Merck, Darmstadt, Germany), a chemical mixture that completely binds oxygen, creating an oxygen-free environment. The bag was sealed and severe hypoxia (<0.5 % O₂) was assessed by color change of a test strip (Anaerotest, Merck) from blue to white within the first 90 min of incubation [16 (link), 17 (link)]. Human umbilical vein endothelial cells (HUVECs) were kindly provided by Dr. Jennifer Esser. Cells were cultured in enhanced endothelial cell growth medium (PELOBiotech GmbH, Martinsried, Germany) and used for angiogenesis assays.

Bacteria were isolated from excrements of dairy cows D, E, and F collected at different time points after Metricycline application (1, 3, and 8 days; Table 1) by the serial plate dilution method. Excrement suspensions of required dilution were prepared from 2 g (ww) of excrement in sterile tap water. Plates with Tryptic soy agar (for aerobic bacteria), Endo agar (for enteric bacteria) (both Difco™; Becton, Dickinson and Co., USA) and Schaedler agar (for anaerobic bacteria; ready-made plates purchased from DULAB s.r.o., Czech Republic), all supplemented with tetracycline (25 mg L⁻¹), were inoculated with 0.1 mL of excrement suspensions diluted respectively to 10⁻⁴, 10⁻³, and 10⁻³. Tryptic soy agar plates were incubated for 7 days at 28°C and Endo agar plates for 7 days at 37°C. Schaedler plates were placed into Anaerobic jars with Anaerocult® A and Anaerotest® (all Merck KGaA, Germany) to maintain and control anaerobic conditions, and incubated for 14 days at 37°C. Colonies of TC-resistant bacteria were streaked onto corresponding fresh plates supplemented with tetracycline to obtain pure cultures. The pure cultures of TC-resistant isolates were transferred to glycerol stocks (2 full bacteriological loops of bacterial biomass, 700 μL of Tryptic soy broth, 300 μL of 50% glycerol) for long term storage at −80°C.