Water jacketed incubator

Helicobacter pylori strains (Table 1) were recovered from frozen glycerol stocks on Brucella broth agar plates, containing 5% fetal calf serum (FCS), in a 9% CO₂-91% air atmosphere at 37°C and 95% humidity in a water jacketed incubator (Thermo Scientific). Liquid cultures were grown in Brucella Broth supplemented with 5% FCS with gentle agitation (120 rpm). E. coli strains DH5α and BL21 (DE3) (Table 1) were grown on Luria-Bertani (LB) agar plates or LB liquid broth with vigorous agitation (250 rpm); when required, ampicillin was added to the medium to achieve a final concentration of 100 μg/ml.

The eventual cytotoxic effects of unsaturated fatty acid vesicles and antioxidant activity of ammonium glycyrrhizinate-loaded nanosystems were evaluated on human keratinocytes (NCTC 2544 cell line). Cells were incubated in plastic culture dishes (100 × 20 mm) in a water jacketed incubator (Thermo Fisher Scientific, Rome, Italy) at 37 °C and 5% CO₂, in presence of minimum essential medium (D-MEM) enriched with glutamax, streptomycin (100 µg/mL)-penicillin (100 µL/mL) solution (1% v/v), amphotericin B (250 µg/mL), and FBS (10% v/v). For cell maintenance, the culture medium was replaced with a fresh medium every 48 h, until ~80% cellular confluence was achieved. The cells were washed with phosphate buffer solution (PBS) and were treated with trypsin/EDTA (1×) solution to induce their detachment. The detached cells were collected in a centrifuge tube and centrifuged (1000 rpm) at room temperature for 10 min with an Eppendorf Centrifuge 5810. Finally, before in vitro experiments, a fresh D-MEM medium was used to resuspend the pellet.

The human breast cancer cell line BCap37 were purchased from the Cell Bank of the Chinese Scientific Academy. The chemoresistant cell lines, Bats-72 and Bads-200, were established by PTX treatment of parental BCap37 cells^{14 (link)}. All cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (31800105, Gibco, Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; 04-0101-1, Biological Industries, Cromwell, CT, USA), and were incubated at 37 °C with 5% CO₂ in a water-jacketed incubator (Thermo Scientific, Waltham, MA, USA). The cell culture medium was changed every 2 days.

Mouse lung epithelial cell line MLE-12, human lung epithelial cell line Beas-2B, and human macrophage cell line U937 were obtained from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MLE-12 cells and Beas-2B cells were cultured in Dulbecco’s modified Eagle’s medium with high glucose (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). U937 cells were cultured in RPMI-1640 medium (Gibco). All medium was supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 mg/mL streptomycin, and 100 units/mL penicillin (Gibco). Cells were incubated in a water-jacketed incubator (Thermo Fisher Scientific) at 37 ℃ in 5% CO₂. To obtain adherent M0 macrophages, U937 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (HY-18739, MCE, Shanghai, China) for 24 h.
For BLM treatment, cells at 70% confluence were washed twice with sterilized phosphate buffered saline (PBS) and cultured in the corresponding medium containing 10 μg/mL BLM for 6 h. For IR treatment, cells were exposed to 6 Gy X-rays (0.833 Gy/min) at room temperature. MLE-12 and Beas-2B cells were divided into four groups: sham-IR + PBS (Ctrl), IR, sham-IR + BLM (BLM), and BLM + IR (B + IR).

Cell line purchased from National Cancer Institute (NCI), Cairo, Egypt and was preserved as “monolayer culture” using RPMI medium supplemented with 10% FBS and 2% Pen/Strep. Cells were incubated at 37 °C in 5% CO₂ in a high humidity atmosphere in a water jacketed incubator (Thermo Fisher Scientific USA). The lines were repetitively sub-cultured to be kept in the exponential growth phase. Sterile conditions were achieved by working under an equipped laminar flow (Microflow Laminar flow cabinet, MDH limited, Hamsphire SP105AA, U.K.). Cells were grouped into control group and treated groups with different concentration (12.5, 25, 50, and 100 µg/ml) of Cis, CoAg, CoAg@Cis. After 48 h, add 10 μl of the MTT reagent (concentration 0.5 mg/ml) to each well. Incubate the microplate for 4 h. Add 100 μl of the Solubilization solution into each well. After complete solubilization of the purple formazan crystals, measure the absorbance of the samples using a microplate (ELISA) reader. The wavelength to measure absorbance of the formazan product is570 nm. The cell viability percentage was calculated using the following equation: $$\text{The cell viability}\left(\%\right)=\left[\text{ODS}/\text{ODC}\right]\times 100.$$ where, ODS stands for the sample’s mean optical density, while ODC is control's mean optical density.