24 well insert

After 24 h transfection, cell concentration was adjusted to 5 × 10⁴ cells/ml with serum-free medium. The upper chamber of Transwell chamber (Costar; 24-well insert, pore size: 8 μ m) was filled with 200 μl of cell suspension, and the lower chamber was filled with 500 μl of medium with 10% FBS. For the invasion assay, polycarbonate filters coated with 50 μl Matrigel (1:9, BD Bioscience) were placed in a Transwell chamber. Cells were incubated for 12 h for the migration assay, and 36 h for the invasion assay. Cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.5 % crystal violet. The migratory cells were visualized and counted in five random visual fields per insert under an inverted microscope at 100× magnification (Nikon MicrophotFX, Japan). The experiment was repeated three times.

After 48 h of transfection, cell concentration in each group was adjusted to 2 × 10⁵ cells/mL with serum-free medium. The upper chamber of Transwell chamber (Costar; 24-well insert, pore size: 8 μm) was filled with 200 μl cell suspension, and the lower chamber was filled with 500 μL of medium supplementing 15 % FBS. For the invasion assay, polycarbonate filters coated with 50 μL Matrigel (1:9, BD Bioscience) were placed in a Transwell chamber. Three wells were used for each group. Cells were incubated for 24 h for the migration assay and 48 h for the invasion assay. Then, the cells on the upper surface were wiped slightly using cotton swabs, and the cells on the lower surface were fixed with 4 % paraformaldehyde and stained with 0.1 % crystal violet. The migratory cells were visualized and counted in five random visual fields per insert under an inverted microscope at 200× magnification (Nikon Microphot-FX, Japan).

For Transwell migration assays (Corning Costar), 24‐well insert, and 8 mm pore polycarbonate membrane were used. The lower chamber was added with 700 ml of media with 20% FBS, while upper insert was added with 1 × 10⁵ cells re‐suspended in serum‐free media post‐transfection. After Transwell membranes were fixed for the set time, they were subjected to crystal violet staining. A light microscope (Olympus) was used for counting the cells adhered to the lower membrane surface.
For wound healing assay, following 24 hr of transfection, a micropipette tip was employed for straight scratch in each well center. In each well, cells migrated to the scratch, which was observed to analyze cell migration. Twenty‐four hours later, the speed of wound closure was measured and normalized to the length at 0 hr. Each assay was conducted in triplicate.

Cell migration assay was conducted using 24-well insert (Costar) with 5-µm pores. PBMCs from normal, healthy donors were activated with either CPG-ODN, a synthetic TLR9 agonist (Invitrogen), or Phytohemagglutinin (PHA) (Sigma) or anti-human IgM (Jackson Immuno Research) and anti CD40 monoclonal antibody, clone B-B20 (BioSite).
The activation status of the cells was then investigated by measuring CD69 expression on CD19+ and CD3+ PBMCs by Flow cytometry prior to migration assay. After 2 days of activation, 0.5 × 10⁶ PBMCs were added in the upper insert of the migration plates whereas 0.6 ml of supernatant from HIV-1 exposed or non-exposed FDCs were added in the lower insert. After 4 h at 37 °C, the inserts were removed, the cells collected from the lower wells and counted in a hemacytometer. Thereafter, the migrated cells were fixed with 2 % PFA and the frequency of CD19 and CD3 positive cells among migrated cells was evaluated.
The monoclonal antibodies (mAbs) used to identify and characterize the PBMCs in the migration assay are PE anti-CD69, FITC and PE anti-CD19, APC and FITC anti-CD3, PE and FITC isotype antibodies all purchased from BD Bioscience.

Migration and invasion assays were performed as described previously [53 (link)]. Briefly, a transwell invasion assay used 2 10⁵ cells plated in a Matrigel (BD Biosciences, Bedford, MA)-coated top chamber and incubated for 24 h (with a 24-well insert and a pore size of 8 μm; Corning Costar, Corning, NY). In this assay, cells which had been pretreated for 1 h with osthole (20~80 μM) were plated in medium without serum or growth factors, and medium supplemented with serum was used as a chemoattractant in the lower chamber. After 24 h of incubation, cells that had invaded through the pores were removed with a cotton swab. Cells on the lower surface of the membrane were fixed with methanol and stained with crystal violet. The number of cells invading through the membrane was counted under a light microscope (×40, three random fields per well).

1 × 10⁵ KYSE‐30 cells, KYSE‐150 cells, or TE‐1 cells transfected with miRNA inhibitors, mimics, or negative controls were pipetted into the upper chambers (24‐well insert, 8 μm pore size; Corning Costar) with 200 μL RPMI‐1640 medium without FBS. Additionally, the lower chambers need to add 600 μL medium with 20% FBS as a chemo‐attractant. After 15−20 h incubation, cells on the upper side were wiped off with a cotton‐tipped swab, while the cells on the lower side were fixed with 4% formaldehyde and stained using 0.05% crystal violet for 10 min. The protocol of cell invasion assays was similar to the cell migration assay. The difference was the 200 μg/mL Matrigel (356234; Becton, Dickinson and Company) need to precoat at the transwell units overnight. The cells on the membrane of the lower side were considered as migrated/invaded cells and were photographed and counted at ×200 magnification.