Minimum essential medium (mem)

One hundred microliters of each virus-positive suspension was inoculated onto confluent monolayers of Aedes albopictus C6/36 (ATCC CRL-1660) or rhesus monkey kidney epithelial (LLC-MK2) (ATCC CCL-7) cells for 1 h in 24-well plates grown in MEM (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, USA). The cultures were incubated at 28 °C (C6/36) and 37 °C (LLC-MK2) in 5% CO₂. Cytopathic effects (CPEs) were monitored and observed daily for the following 6–10 days. The positive isolates were continually propagated in C6/36 cells and LLC-MK2 cells grown in MEM supplemented with 10% heat-inactivated FBS (Gibco, USA) at 28 °C (C6/36) and 37 °C (LLC-MK2) in a 5% CO₂ environment for additional passages. The culture supernatants were harvested for identification and sequencing. Uninfected C6/36 or LLC-MK2 cells were used as negative controls.

Human neuroblastoma cells, SH-SY5Y (ATCC), were grown in 1:1 minimum essential media (MEM) (Gibco by Life Technologies, USA) and nutrient mixture Ham’s F-12 (PAN Biotech, Germany) free of phenol red, supplemented with 1% MEM, non-essential amino acids, 2mM glutamax and 15% fetal bovine serum (Gibco by Life Technologies).

Skin biopsies were obtained using a 3 mm punch from 4 C9orf72 patients and 4 control individuals recruited from the Dermatology Department of the Hospital Clinic of Barcelona. Under sterile conditions, the biopsy was cut and plated at 37 °C with a 5% CO₂ atmosphere in T25 flasks in minimum essential media (MEM) 13% containing 500 mL MEM (Gibco, ThermoFisher Scientific, Madrid, Spain) and 75 mL newborn calf serum (Gibco, ThermoFisher, Madrid, Spain) supplemented with 0.30 mL penicillin (Gibco, ThermoFisher Scientific, Madrid, Spain) and 0.30 streptomycin (Gibco, ThermoFisher Scientific, Madrid, Spain). A trypan blue exclusion test was used to quantify the number cell viability and cell counts using a hemocytometer. No differences in cellular viability were detected when comparing C9orf72 and control cell cultures. All functional assays were performed in cells between passage 5 and 10.

DENV serotype 1 isolated in cell culture from a dengue patient, as previously described,^{12 (link)} was 10-fold serial-diluted using Minimum Essential Media (MEM, Gibco, Thermo Fisher Scientific, Waltham, MA). Three consecutive dilutions (“high titer” = H, “medium titer” = M, and “low titer” = L) were selected based on preliminary testing on RDTs. The dilution “L” was the last dilution with detactable RNA purified from RDT.
Zika virus (H/PF/2013, EVA 001v-EVA1545)– and CHIKV (H20235/STMARTIN/2013, EVA 001v-EVA1540)–inactivated strains provided by the European Virus Archive collection (https://www.european-virus-archive.com/) were 10-fold serial-diluted using Minimum Essential Media (MEM, Gibco, Thermo Fisher Scientific). Following RDT manufacturer instructions for NS1 testing, 100 µL of each virus dilution was loaded on the NS1 cassette of RDTs in triplicate and left for 2 hours for the specimen to dry. Each RDT was then opened, and a 15-mm strip piece was cut out from the sample pad, as described.^{12 (link)}For DENV1, each dilution was loaded onto 39 RDTs, three RDTs were immediately processed (D0), and the rest were divided into three groups and placed at three different temperatures: 4°C, −80°C, and 35°C. After different storage times, 2 days (D2), 1 week (W1), 1 month (M1), and 2 months (M2), three RDTs were taken out from each storage condition and then processed.

Endometrial carcinoma cell lines HEC-1-B, RL-95 and KLE were purchased from ATCC and Ishikawa was purchased from Sigma-Aldrich. Cells were obtained directly from the cell banks, which perform cell line characterizations and passaged in the authors' laboratory for fewer than 6 months after resuscitation. HEC-1-B cell line was maintained in MEM (Gibco) supplemented with 10% fetal bovine serum (FBS), Ishikawa cell line was cultured in MEM (Gibco) supplemented with 5% FBS and KLE and RL-95 were maintained in DMEM (Gibco) supplemented with 10% FBS. All cell lines were maintained with supplementation of 2% penicillin/streptomycin and incubated in humidified chamber at 37°C in 5% CO₂ atmosphere.

The bacterial strains and plasmids used in this work are listed in Table S1 in Supplemental File 1. Single colonies of Escherichia coli bacterial strains grown overnight on Lennox Broth (LB) (43 (link)) agar (1.5% wt/vol) plates or single red S. flexneri colonies grown on trypticase soy agar plates containing 0.03% (wt/vol) Congo red were picked and grown overnight in LB at 37°C for all experiments. Where appropriate, the medium was supplemented with ampicillin (Amp, 100 μg/mL), kanamycin (Kan, 50 μg/mL), streptomycin (Strep, 100 μg/mL), or chloramphenicol (Chl, 25 μg/mL). HeLa cells were routinely cultured and maintained with MEM (Gibco) supplemented with 5% (vol/vol) Fetal bovine serum (FBS), and Madin-Darby canine kidney-2 (MDCK-2) cells were cultured and maintained with MEM(Gibco) or DMEM (Gibco) supplemented with 5% (vol/vol) FBS (Gibco) at 37°C with 5% CO₂.