Penicillin streptomycin solution

L-glutamine and penicillin-streptomycin solutions were purchased from Life Technologies (Grand Island, NY). Concanavalin A (Con A), RPMI 1640 medium, fetal bovine serum (FBS), sodium dodecyl sulfate (SDS), N, N-dimethylformamide (DMF), phosphate buffered saline (PBS), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Vincristine was obtained from Vintec (Columbia, S.A. de C.V., Ciudad de México). Extraction buffer was prepared by dissolving 20% (wt/vol) SDS at 37 °C in a solution of 50% each DMF and demineralized water, and the pH was adjusted to 4.7. The tumor cell line L5178Y-R (mouse DBA/2 lymphoma) was purchased from the American Type Culture Collection (LY-R, ATCC^® CRL-1722™; ATCC, Rockville, MD, USA), maintained in culture flasks with RPMI 1640 medium supplemented with 10% FBS, 1% L-glutamine, and 0.5% penicillin-streptomycin solution (referred as complete RPMI medium) at 37 °C, in a humidified atmosphere of 5% CO₂ in air. Cellular density was kept between 10⁵ and 10⁶ cells/mL.

The hippocampal mouse cells (mHippoE-18, CELLutions Biosystems, Ontario, Canada) were routinely maintained in DMEM high-glucose culture medium (Sigma-Aldrich, Missouri, MO, USA) without sodium pyruvate. The THP1-Blue™ NF-κB cells derived from the human monocytic THP-1 cell line (Invivogen, Toulouse, France) were maintained in RPMI 1640 medium (Cytogen, Zgierz, Poland). Media for both cell lines were supplemented with 10% fetal bovine serum (Biowest, Nuaille, France), 100 U/mL penicillin, and 100 μg/mL streptomycin (Penicillin-Streptomycin Solution ATCC^®). In addition, the medium for THP1-Blue™ NF-κB was supplemented with 100 μg/mL normocin (InvivoGen, San Diego, CA, USA), and 10 μg/mL blastocidin (InvivoGen, USA). The mHippoE-18 cells were trypsinized (Trypsin-EDTA Solution, ATCC^®) twice a week, seeded at a density of 5 × 10⁵ cells per T25 cell culture flask, and incubated at 37 °C and 5% CO₂ to obtain a confluent monolayer. The THP1-Blue™ NF-κB cells were also incubated at 37 °C in a humidified incubator with 5% CO₂ and passaged every 3 days to maintain density < 2 × 10⁶ cells/mL.

Vero (African green monkey kidney) cells were grown in Opti-Pro medium (Invitrogen) supplemented with 50 µg/ml of gentamicin as previously described (39 (link)). For ZIKV infection, Vero cell medium was changed to complete Dulbecco's modified Eagle medium (DMEM [Invitrogen] supplemented with 10% fetal bovine serum [FBS] and 1× penicillin-streptomycin-glutamine solution [Invitrogen]). Mosquito-derived C6/36 cells (Aedes albopictus) and 15P-1 cells (mouse testis Sertoli cells; ATCC) were maintained in complete DMEM at 32°C and 5% CO₂. Primary human foreskin fibroblasts (ATCC) and SH-SY5Y cells (human neuroblastoma, CRL-2266; ATCC) were maintained in complete DMEM at 37°C and 5% CO₂. HTR-8/SVneo human trophoblasts (CRL3271; ATCC) and JAR human placenta cells (HTB-144; ATCC) were maintained in RPMI 1640 medium (ATCC) supplemented with 5% FBS and 1× penicillin-streptomycin solution (Invitrogen) at 37°C and 5% CO₂. BeWo human placenta cells (CCL-98; ATCC) were maintained in Ham’s F-12K medium (ATCC) supplemented with 10% FBS and 1× penicillin-streptomycin-glutamine solution (Invitrogen) at 37°C and 5% CO₂. JEG-3 human placenta cells (HTB-36; ATCC) were maintained in Eagle minimum essential medium (EMEM; ATCC) supplemented with 10% FBS and 1× penicillin-streptomycin solution at 37°C and 5% CO₂.

Cells were transfected using the Lipofectamine CRISPRMAX Cas9 Transfection Reagent (ThermoFisher #CMAX00003) kit. Prior to transfection, A549 lung epithelial cells were plated in 24-well plates at 25% confluency in Dulbecco’s Modified Eagle’s Medium - DMEM (ATCC #30-2002) + 10% Fetal Bovine Serum - FBS (ATCC 30-2020) + 1% Penicillin-Streptomycin solution (ATCC 30-2300) and incubated for 24 h at 37 °C + 5% CO₂. Following incubation, the media was removed and cells were washed with 1× PBS and replaced with fresh DMEM + 10% FBS. Cas9 RNP complexes were formed in a 1:1.2 molar ratio of Cas9 protein to sgRNA with Cas9 Plus reagent to a total volume of 25 μL in Opti-MEM Reduced Serum Medium (ThermoFisher #31985070) per reaction (n = 3). RNPs were added to a mix of 25 μL Opti-MEM I and 1.5 μL CRISPRMAX reagent per reaction, and following a 10 minute room temperature incubation, 50 μL was added to each well. Cells were then incubated at 37 °C + 5% CO₂ for 48 h.

U2OS (ATCC HTB-96) cells were maintained
in high-glucose DMEM, supplemented with 10% fetal bovine serum, 1%
glutamine 200 mM, 1% sodium pyruvate 100 mM, and 1% penicillin–streptomycin
solution (all reagents from Biological Industries, Beit Haemek, Israel),
incubated at 37 °C with 5% CO₂.

Casein sodium salt and acid casein, both from bovine milk, acrylamide (AAm), N,N’- methylenebisacrylamide (MBAAm), ammonium persulfate (APS), N,N,N’,N’ tetramethylenediamine (TEMED), N-(3-aminopropyl)methacrylamide hydrochloride (APMA), lysozyme from chicken egg white (40,000 units/ mg protein), phosphate buffered saline (PBS), NaCl, KCl, NaH₂PO₄, Dulbecco’s modified eagle’s medium (DMEM) (D5796), bovine calf serum, penicillin-streptomycin solution, sodium pyruvate, NIH/3T3 fibroblasts (ATCC^® CRL-1658/Sigma 93061524), trypsin EDTA (ethylenediaminetetraacetic acid) solution, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid solution (MTT solvent), and methanol were all purchased from Sigma (St. Louis, MO, USA). NaHCO₃ was purchased from Panreac (Barcelona, Spain), and NaOH pellets (99%), from Merck (Darmstadt, Germany). Octiset^® was purchased from Schülke (Norderstedt, Germany), and polyhexanide (polyhexamethylene biguanide hydrochloride, PHMB) 94%, from Carbosynth (Berkshire, UK). Mueller–Hinton Agar and Mueller–Hinton Broth were purchased from Oxoid Ltd. (Hampshire, UK). Distilled and deionised (DD, 18 MΩcm, pH 7.7) water was obtained with a Millipore system (Millipore Merck, Darmstadt, Germany).