Dmem ham s f 12 medium

The MTT assay started 24 h after the medium supplemented with nanoparticles was added to 24 well tissue culture test plates seeded with MSC and has been performed every day during the incubation period (n = 3). The culture medium was replaced by 1 ml of 2.5 mg/ml MTT (Sigma, UK) solution prepared in DMEM/Ham`s F-12 medium (Sigma, UK), followed by two hours incubation at 37 °C with 5% CO₂. After incubation, the MTT solution was replaced by 1 ml of 99,8% isopropanol (STANCHEM, Poland). The plates covered with tinfoil have been shaken for 15 min at 100 rpm (ES-20, Biosan), followed by the color change quantification using the plate reader (Synergy H1, BioTek) at 570 nm.
The cell viability was assessed by the formula: $Cellviability\left(\%\right)=\frac{\left({\text{O}\text{D}}_{570}\text{o}\text{f}\text{t}\text{e}\text{s}\text{t}\text{n}\text{a}\text{n}\text{o}\text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}\right)–\left({\text{O}\text{D}}_{570}\text{o}\text{f}\text{b}\text{l}\text{a}\text{n}\text{k}\right)}{\left({\text{O}\text{D}}_{570}\text{o}\text{f}\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)–\left({\text{O}\text{D}}_{570}\text{o}\text{f}\text{b}\text{l}\text{a}\text{n}\text{k}\right)}x100\%$

The NSCLC cell line HCC4006 (HCC) was purchased from ATCC (Manassas, VA, USA). The subline HCC4006^rERLO^0.5, with acquired resistance to erlotinib, was established as previously described [8 (link)] and derived from the Resistant Cancer Cell Line (RCCL) collection (https://research.kent.ac.uk/industrial-biotechnology-centre/the-resistant-cancer-cell-line-rccl-collection/; last access 19 March 2021) [9 (link)]. All cell lines were cultured in DMEM/HAM’s F12 medium (Sigma–Aldrich Chemie GmbH, Munich, Germany) supplemented with 10% fetal bovine serum (Gibco by Thermo Fisher Scientific, BV & Co KG, Braunschweig, Germany) at 37 °C in a humidified 5% CO₂. Medium of HCC4006rERLO0.5 cells was additionally supplemented with 0.5 µM erlotinib.

Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F12 medium mixture (1:1), RPMI-1640 medium, fetal bovine serum (FBS), penicillin/streptomycin solution, nonessential amino acids, l-glutamine, and formaldehyde were from Merck Millipore (Billerica, MA, USA). Trypsin was from VWR International AB (Lund, Sweden). Cell culture plastic, Alexa Fluor^® 594-conjugated goat anti-rat IgG antibody and Alexa Fluor^® 488-conjugated goat anti-rabbit IgG, NHS-fluorescein and propidium iodide (PI) were from Thermo Fisher Scientific (Waltham, MA, USA). Insulin, epidermal growth factor, TPP^® TubeSpin bioreactor tubes, EX-CELL^® chemically-defined serum-free medium for CHO cells, DMEM/Ham’s F12 medium with 15 mM HEPES (2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid) buffer, DMSO (cell culture grade), Hoechst 33342 (bisbenzimide), Mowiol^® 4–88, bovine serum albumin (BSA), β-d-lactose, N,N-dimethylformamide and TWEEN 20 were from Sigma-Aldrich (St. Louis, MO, USA). GPN was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). PD10 columns were from GE Healthcare (Chicago, IL, USA). Recombinant human galectins were produced in E. Coli BL21 Star (DE3) cells and purified by affinity chromatography on lactosyl-sepharose columns, as previously described in Salomonsson et al.^{40 (link)}.

The BeWo cell line has been widely used as an in vitro model for studying trophoblast intercellular fusion and differentiation (Heaton et al., 2008 (link)). BeWo cells (CCL-98, ATCC) were cultured in DMEM/Ham’s F-12 medium (Sigma, St. Louis, MO) containing 10% FBS and 1% penicillin-streptomycin, at 37°C, in an atmosphere of 5% CO₂. BeWo cells (1 × 10⁶ cells/well) were plated in 6 well multi-dishes and maintained in culture until the cells were 70–80% confluent. Differentiation was induced by the addition of 20 µM forskolin (F6886, St. Louis, MO, as positive control) with or without 0.5 µM chemerin, or 0.5 µM chemerin +1 µM α-Neta, in a serum-free medium for 48 h. All experiments involving the cell treatment were accompanied in parallel by vehicle controls. The quantification of BeWo cell fusion under different stimulatory conditions was performed by calculating the fusion index in 20 randomly selected microscopic fields of each condition. The fusion index = number of nuclei in syncytia/total number of nuclei x 100%. The cells were harvested to measure the expression of relevant differentiation markers by qPCR.

The human RPE cell line (ARPE19; ATTC #CRL-2302) and the human telomerase-immortalized RPE cell line (hTERT-RPE1; ATTC #CRL-4000) were cultured in Dulbecco’s Modified Eagle medium (DMEM)/Ham’s F12 medium (1:1 v/v) (Sigma-Aldrich, Saint Louis, MO, USA). Before mixing both mediums, Ham’s F12 was supplemented with 1% of Alanine-Glutamine (Sigma-Aldrich). Human embryonic kidney 293T (HEK293T; ATTC #CRL-3216) cells were cultured in DMEM. Both DMEM/F12 and DMEMs were supplemented with 10% fetal calf serum (Sigma-Aldrich), 1% sodium pyruvate (Sigma-Aldrich) and 1% penicillin-streptomycin (Sigma-Aldrich). The human retinoblastoma cell line (Weri-Rb1; ATTC #HTB-169) was cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Sigma-Aldrich) supplemented with 15% FCS, 2% HEPES (Sigma-Aldrich) and 1% penicillin-streptomycin (Sigma-Aldrich). The cells were grown at 37 °C and 5.0% CO₂.