96 well polypropylene plates

Assays were performed on three Gram-negative bacteria: E. coli (BW25113, Coli Genetic Stock Center), A. baumannii (ATCC 15308, CECT, Valencia, Spain), and Pseudomonas sp. (ATCC15915, CECT). The minimal inhibitory concentration (MIC) of each peptide was determined as described by Wiegand et al. [40 (link)]. Peptide stocks in water were added to polypropylene 96-well plates (Greiner, Frickenhausen, Germany) and serially diluted from 100 to 0.2 μM. Fresh bacteria were incubated at 37 °C in Mueller Hinton Broth (MHB, Condalab, Torrejón de Ardoz, Spain) up to exponential growth and diluted to a final inoculum of 5 × 10⁵ CFU/mL. BSA and acetic acid at 0.04% (w/v) and 0.002% (v/v) final concentration, respectively, were added to avoid peptide self-aggregation. Plates were incubated for 20–22 h at 37 °C. Each peptide was tested in duplicate. MIC values were the lowest peptide concentration where bacterial growth was not detected.

The antibiotic activity was defined by
the minimum peptide concentration where the microorganism is unable
to grow. The assay was done in polypropylene 96-well plates (Greiner,
Frickenhausen, Germany) to avoid peptide binding to plate wells. Peptides
were dissolved in water containing 0.4% w/v bovine serum albumin (BSA)
and 0.02% v/v glacial acetic acid to prevent self-aggregation, following
the reference protocol by Wiegand et al.,^{42 (link)} based on the classical microtiter broth dilution recommended by
the National Committee of Laboratory Safety and Standards (NCLSS).
Initial bacteria inoculum was adjusted to 5 × 10⁵ CFU/mL
in Mueller-Hinton (MH) medium. Incubations were kept at 37 °C
for 24 h. Results are the average of three independent studies. MIC
assays in the presence of serum were performed by incubating the peptide
with human serum at a 1:1 ratio. Then, 10⁶ cfu/mL of Pseudomonas sp. were incubated with serial dilutions of
the peptides for 4 h. To determine the MBC, 50 μL from all wells
were seeded into Petri plates and incubated for 24 h. The MBC concentration
was attributed to the lowest concentration without detectable bacterial
growth for each peptide.

Indirect ELISA was performed in MaxiSorp plates (Nunc) precoated with human H3K27M and H3wt (p14–40) (180 ng per well in PBS). Purified antibodies were added in serial dilutions.
For performance of the competitive ELISA, 96-well polypropylene plates (Greiner) were blocked with 100% FCS for 2 hours at room temperature. Antibody (125 ng) was diluted with H3K27M and H3wt peptide and protein, respectively and incubated overnight at 4°C. Peptide and proteins were 1:2 serial diluted starting from 144 ng. The following morning the mixture was added to a H3K27M (p14–40, 180 ng) precoated MaxiSorp plate.
Plates were washed with PBS supplemented with 0.025% Tween 20 and blocked with 1X ELISA/ELISpot Diluent (eBioscience). Goat anti-human IgG HRP (1:5000, Southern Biotech) was used as secondary antibody. ELISA signal was developed with tetramethylbenzidine (eBioscience) and stopped with 1 M H₂SO_4. Optical density (OD) was measured at 450 nm.