Hts transwell 96 well permeable support

Migration assays with coated or uncoated PCL-co-LC scaffolds were performed as described previously [30 (link)]. In brief, the materials were placed in the bottom chamber of a 96-well Transwell migration plate (Corning^® HTS Transwell^® 96-well permeable supports, 5 µm pore size; Corning) in 250 µL of the migration medium (RPMI 1640 + 2% FBS + 2 mM CaCl₂) and 0.2 ng/µL activated MMP9 in the MMP-activation buffer or buffer without protease were added. In the upper chamber, 1 × 10⁵ Jurkat cells per well were seeded in 100 µL migration medium. Following incubation for 4 h at 37 °C, 100 µL of the cell suspension from the bottom chamber were mixed with 100 µL of trypan blue (0.5% (w/v) in DPBS), incubated for 1 min and counted using Tecan cell counting chips with a Tecan Spark^® multimode microplate reader (Tecan Group, Männedorf, Switzerland). Blank materials were used for data normalization. Migrated cells towards bare materials were set to 1, and the data are presented as x-fold over control as the mean ± standard deviation (SD) from at least three independent experiments.

Embroidered PCL-co-LC scaffolds were kindly provided by Prof. Dr. Stefan Rammelt (TU Dresden, Germany). These scaffolds have an open porosity of 87% with pores from 100 to 800 µm [1 (link)]. The surface was prepared by n-heptane treatment for 10 min three times, and the scaffolds were hydrophilized for 15 min in 50% methanol/1 M NaOH (1:1, (v/v)), and washed with distilled water until the pH was neutral. Coating of PCL-co-LC scaffolds was performed overnight under gentle shaking in a 96-well plate (TPP, Trasadingen, Switzerland) in a concentration-dependent manner or with 1 µM protein solution in 10 mM Tris buffer (pH 8.0) at room temperature. For semi-sterile cell culture handling, the scaffolds were sterilized beforehand two times for 15 min under UV light. The following day, the protein solution was removed and the PCL-co-LC scaffolds were washed either four times with Tris-buffered saline (TBS; 3 g/L tris(hydroxymethyl)aminomethane (Tris), 8 g/L NaCl, 0.2 g/L KCl, pH 7.6) before transferring them into new reaction vessels or three times with Dulbecco´s phosphate-buffered saline (DPBS) before placing them under sterile conditions into bottom wells of a 96-well Transwell migration plate (Corning^® HTS Transwell^® 96-well permeable supports, 5 µm pore size; Corning, Kaiserslautern, Germany).

Migration assays were conducted in 5 μm pore Transwell 96-well plates (Corning HTS Transwell 96 well permeable supports). Here, 5 × 10⁵ MAIT cells in 100 μL serum-free complete medium were seeded in the upper chamber of the Transwell. The bottom chamber was filled with 200 μL of recombinant human chemokines at 200 ng/mL (all from R&D systems) in serum-free complete medium. As positive and negative controls, complete medium or serum-free complete medium were used, respectively. After 3 hours at 37°C, cells in the bottom chamber were stained and counted using FACS counting beads (Countbright Absolute Beads for Flow Cytometry, Thermo Fisher Scientific) according to the manufacturer’s instructions. The migration index was the ratio of the number of cells that migrated in the presence of the chemokine to the number of cells that migrated in the presence of the serum-free medium.