Improved neubauer hemocytometer

A bacterial consortium that was used for the inoculation of plants was formed from a selection of bacterial isolates obtained from the contaminated sediments, as described above. To prepare this consortium, ten bacterial isolates belonging to the phylum Proteobacteria were selected (Table S2). Protobacteria were chosen because they have been identified as a dominant bacterial group within the same contaminated sediments in previous studies [38 (link),39 (link)]. Selected bacterial isolates were individually subcultured in impoverished Tryptic Soy Broth (TSB, 3 g/ L) at 27 °C for 72 h, after which the liquid cultures were centrifuged at 5000 rpm for 10 min at 4 °C. The supernatant was then discarded, and the resulting bacterial cell pellet was re-suspended in 1 L of a sterile isotonic 0.154 M NaCl solution. Cell counts for each isolate were then performed using a Neubauer improved hemocytometer (Sigma-Aldrich, Oakville, ON, Canada), and the inoculum was made up by suspending all the selected isolates in equal numbers in a final volume of 2.4 L at a final concentration of 2.4 × 10⁹ CFU/mL.

Cell viability was assessed by evaluating the membrane integrity using the Evans blue method. Therefore, viable cells (unstained) and dead cells (stained) were identified and counted on a Neubauer improved hemocytometer (Sigma-Aldrich) using an optic microscope (Zeiss Axio Imager Z1). The cell viability was defined as the percentage of viable cells relative to total cells (i.e., viable and dead cells). Cell growth rate was assessed every week using the cells counts with Evans blue method, as previously described in Ventura et al. (2018) (link) and Fricano et al. (2020) (link). The following formula was then used to calculate the weekly growth rate:

Briefly, 1 × 10⁴ cells were plated into 96-well plate for 24 h and serum-starved for additional 24 h, before treated with complete media, control media (media containing 1% FBS) or fibroblast conditioned media (1.0 μg/μl) with or without chemicals, recombinant proteins or neutralizing antibodies (BioLegend, CA, USA) for subsequent 24-72 h. At the end of treatment, MTT solution (5 mg/ml) was added into each well, followed by 4 h incubation at 37°C. The formazan crystals were dissolved with sodium dodecyl sulfate before reading the absorbance using CHAMELEON™V plate reader (Hidex, Finland) at 570 nm with reference wavelength of 630 nm.
To measure cell proliferation, we determined BrdU incorporation into the cellular DNA using an ELISA-based approach (Cell Signaling Technology, MA, USA). Cells were seeded and treated as described above, and at the end of treatment, BrdU solution was added for 24 h. Following fixation and denaturation, the cells were labeled with antibodies and washed before added with TMB substrate. Color development was terminated with a STOP solution, and measured at 450 nm using CHAMELEON™V plate reader. In addition, the cells were also stained with 0.04% trypan blue to count for number of viable cells, using neubauer improved hemocytometer (Sigma Aldrich).