Flat bottom 24 well plate

Soft agar was carried out by growing 2, 000 cells in 0.4% bactoagar containing 10% FBS on a bottom layer of solidified 0.8% bactoagar (Sigma, Shanghai, China) with 10% FBS in a 24-well flat-bottom plate (Corning) as our previously described [26 (link)]. After incubation for 2–3 weeks at 37 °C in a humidified incubator, the colonies (containing ≥50 cells) were counted with an inverted optical microscope.

A primary testis tissue culture system was used to study the effects of hINSL3 on germ and somatic cell proliferation, androgen release and testicular mRNA levels, according to protocols previously established (Leal et al. 2009b ). The concentration of 100 ng hINSL3/ml was chosen based on a pilot experiment and data published in mammals (Pathirana et al. 2012 (link)). To study proliferation and transcript levels, the two testes from each fish were dissected and incubated for 7 days, one under stimulatory conditions (receiving medium containing hINSL3) and the other under basal conditions (receiving only tissue culture medium). The testes were placed on a 0.25-cm² piece of nitrocellulose membrane (25 μm thickness, 0.22 μm pore size; Millipore, Billerica, Mass., USA), on top of a 700-μl agarose cylinder (1.5 % [w/v] in Ringer’s solution, pH 7.4) that was placed in a 24-well flat-bottom plate (Corning, New York, USA). Medium (1 ml) was added such that the agar cylinder was just not submerged; the medium was refreshed after 4 days. To study androgen release, testes were submerged in 200 μl tissue culture medium in a 96-well plate for ~18 h. All components for the tissue culture studies were freshly prepared according to published protocols (Leal et al. 2009b ).

An overnight culture (1 ml) of S. gordonii SecCR1 grown stationary in HTVG under anaerobic conditions (GasPak, anaerobic gas generating system, BD) was added to 9 ml of pre-warmed HTVG, and the culture was incubated at 37°C under anaerobic conditions. A second overnight culture (1 ml) grown stationary in HTVG in a CO₂ incubator was added to 9 ml of pre-warmed HTVG, and the culture was incubated aerobically in ambient atmosphere at 37˚C on a shaker (20 rpm). When the cultures reached OD₆₀₀ = 0.8, cells were harvested by centrifugation and analyzed by western blotting for MsrAB, SdbB, and Sgo_1177.
To test the level of expression of MsrAB, SdbB, and SdbC in biofilm and planktonic cells, overnight cultures of S. gordonii SecCR1 grown in HTVG were centrifugation (3000 x g for 10 min), and the cells were suspended in biofilm medium to an OD₆₀₀ = 0.25. The cell suspension (1 ml/well) was used to inoculate a 24-well flat-bottom plate (Costar). The plates were incubated at 37°C, 5% CO₂. Next day, the supernatant containing the planktonic cells was collected, and the wells were washed once with 1 ml of sterilized PBS to remove loosely attached cells. The biofilm cells were resuspended in biofilm medium (1ml/well) by vigorous pipetting. The planktonic and biofilm cell suspension were adjusted to OD₆₀₀ = 0.8 and cells were then harvested by centrifugation and analyzed by western blotting.