Flat bottom 96 well plate

Adherent BMDM or BMDC were harvested using cell scraper, 0.5 × 10⁵ cells/well were plated into 96-well flat-bottom plates (Sarstedt, Nuembrecht, Germany) and pre-stimulated with 500 ng/ml lipopolysaccharide (LPS, Sigma-Aldrich). 24 h thereafter, cells were incubated for 2 h with DyLight-405 labeled conformational mMOG (mMOG-DyLight-405) or ovalbumin (OVA)-FITC (Life Technologies, Thermo Fisher Scientific) in the presence of 50 µg/ml 8.18C5 Ab, 8.18C5 F(ab′)₂ fragment, anti-OVA Ab (clone: TOSG1C6, BioLegend, San Diego, USA) or isotype control Ab (clone: MOPC-21). To investigate phagocytosis of conformational hMOG, murine BMDM which are known to recognize human IgG via their Fc receptor [37 (link)] were incubated with hMOG-DyLight-405 for 2 h in the presence of 15 µg/ml of the respective IgG preparation. Where indicated, Fcγ receptors were blocked using anti-mouse CD16/CD32 Ab (Clone: 2.4G2, 1:100 dilution).

3000–4000 HT-29 cells/well were seeded in 96-well flat bottom plates (Sarstedt, Germany) coated with 50 μl 0.15% agarose (Sigma Aldrich, United States) and incubated for 72 h. Established spheroids [mean spheroid starting sizes of 0.030 mm³ ± 0.005 (SD) mm³] were treated with (a) 100, 250 or 400 nM seeMet 12 monotherapy, (b) 2 and 5 μg/ml sorafenib monotherapy and the combination of sorafenib and 100 and 250 nM seeMet 12 or (c) 2, 4, and 6 Gy radiation and the combination with 100 and 250 nM seeMet 12. Twice a week, half of the incubation medium was replaced with fresh medium. Spheroids were followed for >14 days and pictures were taken 3–4 times a week using an inverted microscope Nikon Diaphot (Nikon, Japan) mounted with Canon EOS 700D camera (Canon Inc., Japan). Spheroid sizes were measured and analyzed using ImageJ 1.51k software (NIH, Bethesda, MD, United States). Irradiation of the multicellular tumor spheroids was performed using X-ray irradiation with a Linear accelerator “Elekta Precise Treatment System” at the unit for radiotherapy treatment (Strålbehandlingsavdelningen) at Uppsala University Hospital. The dose rate was 5 Gy/min.

All L-form strains were cultured in liquid L phase broth (LPB) and solid L phase medium agar (LPMA). LPB consists of a 1:1 mixture of yeast extract malt extract (YEME) and tryptic soy broth supplemented with 10% sucrose (TSBS) and 25 mM MgCl₂. LPMA consists of LPB supplemented with 1.5% agar, 5% horse serum, and 25 mM MgCl₂ (9 ). P-buffer containing sucrose, K₂SO₄, MgCl₂, trace elements, KH₂PO₄, CaCl₂, and N-tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid (TES) (9 ) was used for transformation and all fusion experiments and was supplemented with 1 mg/mL DNase I (Roche Diagnostics GmbH). The antibiotics apramycin (Duchefa Biochemie) and hygromycin (Duchefa Biochemie) were used for selection and were added at final concentrations of 50 μg/mL and 100 μg/mL, respectively. Growth conditions for all cultures were 30°C in an orbital shaker (New Brunswick Scientific Innova) with 100 rpm for the liquid cultures. Centrifugation (Eppendorf centrifuge 5424) conditions were always 1,000 × g for 10 min (<1 mL) or 30 min (>10 mL), depending on culture volume. The above-mentioned culture conditions and centrifugation settings were applied throughout the study unless mentioned otherwise. All measurements for optical densities (ODs) of samples were done with 200-μL aliquots of culture in a 96-well flat-bottom plate (Sarstedt) using the Tecan Spectramax plate reader.

All L-form strains were cultured in liquid L phase broth (LPB) and solid L phase medium agar (LPMA). LPB consists of a 1:1 mixture of yeast extract malt extract (YEME) and tryptic soy broth supplemented with 10% sucrose (TSBS) and 25 mM MgCl₂. LPMA consists of LPB supplemented with 1.5% agar, 5% horse serum, and 25 mM MgCl₂ (9 ). P-buffer containing sucrose, K₂SO₄, MgCl₂, trace elements, KH₂PO₄, CaCl₂, and N-tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid (TES) (9 ) was used for transformation and all fusion experiments and was supplemented with 1 mg/mL DNase I (Roche Diagnostics GmbH). The antibiotics apramycin (Duchefa Biochemie) and hygromycin (Duchefa Biochemie) were used for selection and were added at final concentrations of 50 μg/mL and 100 μg/mL, respectively. Growth conditions for all cultures were 30°C in an orbital shaker (New Brunswick Scientific Innova) with 100 rpm for the liquid cultures. Centrifugation (Eppendorf centrifuge 5424) conditions were always 1,000 × g for 10 min (<1 mL) or 30 min (>10 mL), depending on culture volume. The above-mentioned culture conditions and centrifugation settings were applied throughout the study unless mentioned otherwise. All measurements for optical densities (ODs) of samples were done with 200-μL aliquots of culture in a 96-well flat-bottom plate (Sarstedt) using the Tecan Spectramax plate reader.

Glycoside
hydrolases from the GH1 family (Lp_0440, Lp_0906, Lp_1401, Lp_2777,
Lp_2778, Lp_3011, Lp_3132, Lp_3512, Lp_3525, Lp_3526, and Lp_3629
proteins) and GH42 family (LacA or Lp_3469) from L.
plantarum WCFS1 were produced as previously described.^{13 (link),20 (link)} The hydrolytic activity of these 12 glycoside hydrolases was determined
by using a library of 24 pNP-glycoside derivatives.^{13 (link)} The assay was performed in a 96-well flat bottom
plate (Sarstedt), where each well contained a different substrate
(10 mM). Briefly, the reaction consisted of 4 μg protein in
50 mM MOPS buffer pH 7.0 containing 20 mM NaCl and 1 mM DTT. The reaction
was incubated at 30 °C for 10 min and stopped by the addition
of 1 M sodium carbonate at pH 9.0. Hydrolysis of each pNP-glycoside derivative was colorimetrically measured by liberation
of p-nitrophenolate (pNP) at 420
nm in a microplate spectrophotometer PowerWave HT (Bio-Tek, USA).
Controls without the enzyme for evaluating spontaneous hydrolysis
of the tested substrates were carried out. Experiments were performed
in triplicate, and results were expressed as an average activities
± standard deviation.

The proliferation of hFOB 1.19 cells was evaluated using a WST-1 assay kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol. Briefly, the cells in a 24-well plate were incubated with the extract or fresh medium for 48 h, and then treated with 40 µL of WST-1 reagent, followed by 2 h incubation at 34 °C and 5% CO₂ in a CB240 incubator (Binder, Tuttlingen, Germany)
The media were collected from each well, and transferred to 96-well flat bottom plate (Sarstedt, Nümbrecht, Germany). The optical density at 450 nm and 620 nm was measured using a plate reader, Epoch (BioTek Instruments, Winooski, VT, USA). The untreated cells and blank were included into each assay. The values of background and blank absorbance were subtracted from all the absorbance values. The percentage of proliferation was calculated by the equation below:
The control absorbance was calculated as a mean of triplicate.