Hank s solution

Lamina propria macrophages from the intestinal mucosa of septic mice were isolated as previously reported^{49 (link), 50 (link)}. Briefly, the tissues were first predigested to eliminate epithelial cells. The intestinal tissues were then digested with digestion solution (1 mg/mL collagenase type VIII + 3 mg/mL dispase II + 40 μg/mL DNase I + 5% FBS) + Hanks Solution (all reagents were from Sigma-Aldrich, St Louis, MO, USA). After incubation at 37 °C for 30 min while shaking (150 rpm), the tissues were filtered through a 100-μm nylon mesh filter (Fisher brand, Loughborough, UK). The remaining pieces were repeatedly digested and filtered, and the filtered suspensions were rinsed with PBS. The cell suspensions were centrifuged, resuspended in fresh PBS, and filtered again through a 40-μm nylon mesh filter (Fisher brand, Loughborough, UK).

Intracellular ROS level was measured using the fluorescent dye, dichlorofluorescein diacetate (H2-DCF-DA), and a nonpolar compound converted to a polar derivative (dichlorofluorescein) by cellular esterase after incorporation into cells [21 (link)]. RLECs (5 × 10³) were cultured in 96-well plates for 120 hrs with DMEM and 5% FBS; 5.5 mM, 55 mM D-glucose, or 55 mM D-mannitol; and 50 μg/mL purified honey or 5 or 50 μg/mL propolis.
The medium was replaced with Hank's solution (Sigma) containing 10 μM DCFH-DA (Cayman Chemical, Ann Arbor, MI) and incubated for 10 min at room temperature. Intracellular fluorescence was detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using Spectra Max Gemini EM (Molecular Devices, Sunnyvale, CA).

Decamethonium bromide (DMB) (Sigma, France) and pancuronium bromide (PB) (Sigma) solutions were freshly prepared before each experiment at 12 mM or 11 mM, respectively, in Hank’s solution (Sigma) with Penicillin-Streptomycin at 1% (Gibco, France). The control solution was prepared using Hank’s solution with 1% of Penicillin-Streptomycin. 100 µl of DMB, PB or control solutions were administrated in chick embryos at E7.5 and E8.5. Embryos were fixed at E8 for the 12 hr time point, at E8.5 for the 24 hr time point and at E9.5 for the 48 hr time point.

In order to confirm that Fam114A1 is related to the activity of melanocytes, we compared the expression of Fam114A1 in the melanocytes of normal individuals (MC) and patients with vitiligo (MCV). Vitiligo is a typical skin disease characterized by impaired melanocyte function [12 (link), 26 (link)–28 (link)].
Skin specimens for melanocyte culture were provided by the Third People’s Hospital of Hangzhou. Methods for isolating and cultivating cells from the specimens were previously described [29 (link)–32 (link)]. Specimens were washed twice with Hanks' solution (SIGMA, USA), then digested with 0.25% trypsin solution and 0.02% EDTA(SIGMA, USA) at 37° C for 30min, and cells were separated under an anatomical microscope. Suspension cells in F12 medium (GIBCO) containing 20 ng/mL bFGF(Pepro Tech), 20 ng/mL IBMX(SIGMA), 10 ng/mLCT(SIGMA), 50 ng/mL Gentamicin (GIBCO) and 10% fetal bovine serum (GIBCO) were placed in culture flask. The cells were cultured in a 5% CO2 incubator. After 3 days, geneticin (SIGMA) was added in the culture medium to eliminate the contamination of fibroblast and keratinocyte. The research protocol was approved by the Ethics Committee of Hangzhou Third People's Hospital.

For the click staining, 5 × 10⁶ PBMC were washed once with HBSS (Hanks Solution, Sigma Aldrich). Pellets were re-suspended in 100 µl of HBSS containing 25 µM ω-N₃-C6-ceramide (6-azido-N-((2S, 3 R, E)-1,3-dihydroxyoctadec-4-en-2-yl)hexanamide)²⁷ and then further processed as described earlier.²⁸ (link)^,29 After the final three washing steps with HBSS, cells were immediately analysed by FACS.

MDR GC cells were treated with a combination of Ubenimex and the pEGFP-N1-CD13 plasmid for 24 h and then exposed to FOLFOX. At 24 or 48 h later, cells were collected and suspended in Hanks solution (Sigma-Aldrich), followed by incubation with fluorescent-labeled Cyto-C antibodies (2 μL) at 4°C for 1 h. All stained cells were analyzed with a flow cytometer, and the data were processed with WinMDI 2.9 software (Scripps Research Institute, La Jolla, CA).