Diff quick stain

The cellular effects of knockdown of PD‐L1 were further investigated in FaDu and SCC‐9 cells using the BD Biosciences BioCoat control chamber and Matrigel invasion chamber. 1 × 10⁵ cells were transfected with siRNA1/siRNA2 or siRNA scramble control, or si‐circ_0000052 or si‐circ negative control, and plated on either the 6‐well control inserts (PET membrane) or trans‐well chambers pre‐coated with Matrigel. A medium containing 15% foetal bovine serum in the lower chamber served as the chemoattractant. After 24 h incubation, non‐migrating or invading cells were removed from the upper surface of the membrane with cotton swabs. The migrating or invasive cells attached to the low surface of the membrane insert were then fixed and stained with Diff‐Quick Stain (BD Biosciences). The number of migrating or invasive cells was counted under a microscope.

The migration and invasive ability of MDA-MB-231 cells were assessed using the BD Biosciences BioCoat Control Chamber and the Matrigel Invasion Chamber. 1 × 10⁵ cells were transfected with either antagomiR-224 or NC and plated on either control inserts (PET membrane) or transwell chambers precoated with Matrigel. The medium containing 15% fetal bovine serum in the lower chamber served as the chemoattractant. After 24 hours' incubation, noninvading cells were removed from the upper surface of the membrane, with cotton swabs. The invasive cells attached to the lower surface of the membrane insert were then fixed and stained with a Diff-Quick Stain (BD Biosciences). The number of invasive cells was counted under a microscope.

Cell invasion was determined using BD BioCoat Tumor Invasion Assay System (BD Biosciences) according to the instructions of the manufacturer. Briefly, 4×10⁵ of 60As6 cells with serum-free media were seeded into the upper chamber of the system. Bottom wells were filled with media with 10 ng/ml SDF-1β (Invitrogen, Carlsbad, CA). After 24 hr incubation, the cells in the upper chamber were removed and the cells that invaded through the Matrigel membrane were stained with Diff-Quick stain (BD Biosciences) and counted manually.