48 well microchemotaxis chamber

Migration assays were performed in 48-well micro chemotaxis chambers (Neuroprobe) through Collagen I-coated polycarbonate membranes with 8μm pores (Neuroprobe). The bottom chambers were filled with 30 μl of complete media per well with/without the tested compounds. HUVECs were harvested and resuspended at 10⁶ cells/ ml in complete media. The upper chambers were loaded with 50 μl cell suspension and incubated for 6 h at 37°C. Next, the filters were fixed with methanol and stained with hematoxylin solution. The cells that had migrated through the filter were manually quantified under an inverted microscope at 20X magnification.

HPASMCs, HPMVECs, U937 pre‐treated with PMA¹⁰ and RAW264.7 cell chemotaxis were assessed using the 48‐well micro chemotaxis chambers (Neuroprobe, Inc., Gaithersburg, MD, USA), as previously described.¹¹

Migration assays were performed using a 48-well microchemotaxis chamber (Neuroprobe, Cabin John, MD) as described previously (9 (link)). Wt or mIGFBP-6 (1 μg/ml) or IGF-II (100 ng/ml) in SFM were added to the bottom well of the chamber. Polycarbonate filters (12 μm pore size, coated with 100 μg/ml gelatine in 10 mM acetic acid at room temperature for 30 min) were placed over the bottom wells, and cells (5 × 10⁴ cells/well) in SFM were seeded in the top wells. In some experiments, pathway inhibitors PD98095 or SP600125 (30 μM) in SFM were added to both bottom and top chambers. After 16 h, the filter was removed, fixed with 100% methanol for 5 min, and stained with 0.5% crystal violet, 50% methanol for 20 min. The filter was then washed in water to remove excess dye and unbound cells. After mounting the filter on a glass slide, non-migrated cells on the top side of the filter were completely removed. Three random fields per well were photographed, and migrating cells were counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA)¹. Each data point represents four to seven replicates.

Cell migration was assessed in a 48-well microchemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA) as described previously [19 (link)]. The chamber consisted of acrylic top and bottom plates, each containing 48 matched wells. Twenty-six microliters of FBS-free medium containing tested substance (10 μg/ml) were filled in wells of the bottom plate. Wells filled with medium containing 0.0001 % of acetic acid served as control. Subsequently, the bottom plate was covered with a polycarbonate filter with 8-μm pore size (Neuroprobe, Gaithersburg, MD, USA) and the top plate was applied so that each well corresponded to that of the bottom plate. 1 × 10⁴ cells resuspended in 50 μl FBS-free medium were added to each well of the top plate and the whole chamber was incubated at 37 °C in humidified air with 5 % CO₂ for 8 h. After incubation, cells on the upper surface of the filter were removed over the wiper blade and the filters were then fixed with methanol and stained using Hemacolor staining kit (Merck, Darmstadt, Germany). The cells migrated across the filter were counted under a light microscope at high-power magnification (×100) to measure transmigration in each well. Four fields were counted in each well and the total number was calculated. Four wells were used for each group; experiments were repeated in triplicate.

To evaluate the effect of asprosin on the release of chemotactic factors responsible for the recruitment of immunocompetent cells, a 48-well microchemotaxis chamber (Neuro Probe) and the RAW 264.7 macrophage cell line were used. The test was performed according to the manufacturer’s protocol. N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) was used as a chemoattractant agent in the positive control sample, and DMEM medium was used as the negative control. The chamber was incubated for 45 min under standard culture conditions. The membrane was subsequently washed in PBS and stained. The macrophages that had migrated to the lower side of the membrane were counted in four microscopic fields in each well. The results are expressed as the mean numbers of migratory macrophages per well.