96 well tissue culture plate

Peptide toxicity was evaluated by using human red blood cells (hRBCs) and other host cells. Hemolysis was conducted as described elsewhere [28 (link)]. Briefly, hRBCs, obtained from the UNMC Blood Bank, were washed three times with phosphate-buffered saline (PBS) and diluted to a 2% solution (v/v). After peptide treatment, incubation at 37 °C for one hour, and centrifugation at 13,000 rpm, aliquots of the supernatant were carefully transferred to a fresh 96-well microplate. The amount of hemoglobin released was measured at 545 nm. The percent lysis was calculated by assuming 100% release when human blood cells were treated with 2% Triton X-100, and 0% release when incubated with PBS buffer. The peptide concentration that caused 50% lysis of hRBCs was defined as HC₅₀.
Vero cells (ATCC, CCL-81) or Calu-3 (ATCC, HTB-55) cells were seeded into 96-well tissue culture plates (Greiner Bio-One, Monroe, NC) and treated with different concentrations of peptides for 24/48 h at 37 °C. Cell viability was examined by Vybrant^® MTT Cell Proliferation Assay Kit (Thermo Fisher Scientific, Grand Island, NY) following the manufacturer’s instructions. Toxicity assays using HaCaT cells were conducted similarly as described elsewhere [34 (link)].

D. discoideum cells were grown to 80% confluency (~1 x 10⁵ CFU/well) in HL/5 medium in 96-well tissue culture plates (Greiner Bio-One) at 21°C. 10⁵ CFU of bacteria in 10 μl were added to each well, which corresponds to a multiplicity of infection (MOI) of 1. Plates were centrifuged at 300 x g for 10 minutes at room temperature and then incubated at 21°C. After 45 minutes, the supernatants of wells were replaced with 100 μl of 300 μg/ml gentamicin solution (Sigma-Aldrich) in HL/5 to kill extracellular bacteria, and plates were incubated at 21°C. At 2 hours post gentamicin treatment, the wells were washed three times with PBS to remove the antibiotic. To lyse the amoebae, 100 μl of 0.1% Triton-X solution was added, followed by a 5-minute incubation and subsequent vigorous pipetting and vortexing. To enumerate the intracellular bacteria, serial dilutions of the lysates were plated onto BG agar plates, and colony numbers were counted after incubation at 37°C for two to four days. As control, similar numbers of each species were cultured with media containing gentamicin without amoebae. After 2 hours, serial dilution of the solution were plated on BG agar for bacterial enumeration.

Cell fusion was performed according to the classical method [26] , [27] . Following the last I.P. injection, the mouse with the highest anti-A, anti-B, and anti-E serum titers was boosted with an intravenous (I.V.) injection of a soluble trivalent (10 µg HcA +10 µg HcB +10 µg HcE) vaccine preparation. Three days after the I.V. injection, the mouse was euthanized and its splenocytes were fused with exponentially growing myeloma cells using 50% polyethylene glycol (PEG) 1500 in EnDMS. Following cell fusion, the cells were suspended in HAT selection medium together with splenocytes from a naïve non-immunized mouse that was used as a feeder layer. The cells were dispensed into 96-well tissue culture plates (Greiner, Austria) and incubated for 5–10 days at 37°C in 5% CO₂ before screening for secreted antibodies.

Cells were counted using trypan blue (Biological Industries), and 10⁵ cells/well were placed in 24-well tissue culture plates (Greiner Bio-One, Germany) with 0.5 mL of growing medium, or 10⁴ cells/well were placed in 96-well tissue culture plates (Greiner Bio-One, Germany) with 100 µl growing medium. MC3 or EA-PIP LNPs were added to the wells at RNA amounts of 0.06 to 1 mg/ mL. Cells were incubated with the LNPs under standard culture conditions for up to 48 h. Then, cells were washed three times, incubated in fresh culture medium, and were taken for functional Luciferase assay system (Promega), XTT cell proliferation assay (Biological Industries) or FACS analysis for EGFP expression or stained with APC Annexin V (BioLegend) and propidium iodide (SigmaAldrich) according to the manufucturers' recommendations and then analyzed by FACS (Cytoflex, Beckman Coulter, USA. 2X10⁵ cells per FACS measurement).

Cytotoxicity was assessed by LDH release from 16HBE-14o- cells. Cells were seeded in 1x MEM supplemented with 10% FBS (Corning) at a density of 2.5 × 10⁴ in a 96-well tissue culture plates (Greiner) in triplicate and grown at 37 °C with 5% CO₂ for 48 h. Bacteria were grown for 20 h at 37 °C with 5% CO₂ and resuspended in 3 mL of HI broth to on OD₆₀₀ ~ 0.8. After resuspension, the culture media for the 16HBE-140- cells was replaced with 100 μL of prewarmed, serum-free 1x MEM. A 5 μL aliquot of the resuspended culture was added (M.O.I. ~100), and the plates were centrifuged for 2 min at 1000 x g followed by incubation for 1 h at 37 °C with 5% CO_2. The infected cells were once again centrifuged for 2 min at 1000 x g, and 50 μL of supernatant was removed for quantification. LDH in the cell supernatants was quantified using the Cytotoxicity Detection kit (Roche), following manufacturer’s instructions. Plates were developed for 40 min in the dark, and the absorbance at 490 nm was quantified on a plate reader (PerkinElmer). Percent maximum LDH release was calculated for each strain relative to a 1% Triton X-100 lysis control. Statistical analysis was performed in Graphpad Prism v.9.2.0

The Hep-2, BGM, MA104, and CaCo-2 cell cultures were each seeded at a concentration of 2 × 105 cells/mL and grown individually in 96-well tissue culture plates (Greiner-Bio one, Frickenhausen, Germany). After an incubation period of 24 h at 37 °C in an atmosphere containing 5% (v/v) CO₂, cell monolayers were confluent. At this point, the media was withdrawn from each well and replaced with 100 μL of bi-fold dilutions of the sample examined that were prepared in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO BRL, Darmstadt, Germany). For the purpose of cell controls, 100 μL of DMEM containing no samples was added. All the cultures were kept in an atmosphere with 5% (v/v) CO₂ that was humidified and incubated at 37 °C for a total of 72 h. Daily observations showed that the cells had lost their confluence, were rounding and shrinking, and that the cytoplasm was granulating and vacuolizing. Morphology shifts were observed and scored [33 (link)].