Model 3550 microplate reader

Cell viability was evaluated by CCK-8 assay. Briefly, BMCs were plated in 96-well culture plates (1×10⁵ cells/well) with RPMI-1640 supplemented with 10% FBS. Subsequently, cells were divided into different groups treated with various concentrations of the herbal aqueous extract (0, 10, 50, 100, 250, 500, 750 and 1000 μg/mL) at 37°C in a humidified 5% CO₂ incubator. After 24 h of incubation, CCK-8 solution (20 μL) was added into each well and then incubated for another 4 h at 37°C. The optical density of the purple solution was measured at 570 nm by a microplate reader (Model 3550 Microplate Reader, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The results of the CCK-8 assay indicated that the two concentrations (100 μg/mL and 250 μg/mL) of freeze-dried powder were suitable for cell stimulation. In particular, the two concentrations were optimal for reflecting the dependence and correlation between drug intervention time and drug dose (S1 Fig).

SDF-1α was measured in CM from MCF-7 and MDA-MB-231 cells using a commercially available ELISA Kit in accordance with the instructions by the manufacturer (Human CXCL12/SDF-1 alpha Quantikine ELISA Kit, R&D Systems, Inc. Minneapolis, USA).
For binding assay, breast cancer cells were untreated (-) or treated with BRL 10 μM in phenol red-free media containing 5% CT-FBS for 24 h. Then, cells were harvested with versene reagent, washed twice in PBS and 10³ cells/well were incubated with CAF-CM in a final volume of 100 μl binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 150 mM NaCl, 5 mM MgCl2, 5% bovine serum albumin). Samples were incubated for 60 min at 4°C with rotation. After incubation, cells were centrifuged and washed twice with 300 μl wash buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 500 mM NaCl, 5 mM MgCl2) and freezed to −20°C and thawed to room temperature 3 times and then centrifuged at 1500×g for 10 minutes at 2 − 8°C to remove cellular debris. The supernatants were collected for assaying human SDF-1α levels (R&D Systems). The optical density of each well was determined using a microplate reader at 450 nm (Bio-Rad Model 3550 microplate reader, Richmond, CA) and normalized for cell number. At least three independent experiments were performed.

A total of 1 × 10⁴ cells were plated in 96-well flat-bottom plates in 100 μL of medium. The next day, cells were exposed to the antidiabetic drugs and potential herbal ingredients at different concentrations. After one day from the last drug addition, 20 μL of 5 mg/mL MTT solution in PBS was added to each well for 4 h. The medium was removed, and 200 μL DMSO was added to each well to dissolve the formazan crystals. Absorption at 570 nm was determined using a Bio Rad microplate reader (Model 3550 microplate reader, Bio-Rad Laboratories, Richmond, CA). Triplicate wells were assayed for each condition, and standard deviations were determined. The concentrations of each agent with survival rates >90% were selected for use in the following assays.

Neurospheres were dissociated into single cells by Accutase and resuspended in PBS. The cells were seeded at a density of 1 × 10⁵ cells/mL in nine culture plates. The growth rates of the cells were then determined by CCK-8 assay. Next, cells were incubated in 10 μL CCK-8 working solution at 0, 24, 48, and 72 h, followed by incubation for 2 h at 37°C. The absorbance was finally measured at 450 nm using a model 3550 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).