Transwell inserts

MRC1^+/Hi (CD11b⁺Ly6G⁻F4/80⁺MRC1^Hi) and MRC1^−/Lo (CD11b⁺Ly6G⁻F4/80⁺MRC1^Hi) TAMs were FACSorted from dissociated cyclophosphamide-treated LLC1 tumors and seeded into Transwell inserts (4 μm pores, VWR International), 50 × 10³ cell per well. Lower chambers contained either medium alone, medium supplemented with 10 nmol/L BSA or 10 nmol/L recombinant murine CXCL12 (100 ng/mL). After an incubation of 6 hours at 37°C, the chambers were dissassembled and the membranes stained with crystal violet. The upper surface of each Transwell insert was scraped to remove non-migrated cells before quantification.

For the scratch wound assay, approximately 5 × 10⁵ TNBC cells were plated on 6-well plates for 24 h. A single straight scratch was made in the monolayer with a small pipet tip. After washing the detached cells with PBS, the cells were transfected with therapeutic and control nanoparticles and monitored for up to 48 h, or until the scratch wounds were closed. Images were taken using the Moticam T2 camera.
For Transwell migration assays, 1–2 × 10⁵ cells transfected with nanoparticles were plated on Transwell inserts (VWR, Radnor, PA) coated with 0.28 mg/mL Corning™ Matrigel™ Membrane Matrix (Corning, NY). The next day, the un-migrated cells in the inserts were removed by swabbing with Q-tips. The migrated cells were fixed by treating the inserts with 10% formalin for 10 min. After washing with PBS three times, the inserts were placed in 0.05% crystal violet stain for 20 min to stain the migrated cells. The inserts were washed under running water to remove excess stain and set to dry overnight. Images of the purple migrated cells were taken using the Moticam T2 camera. The cells in the images were counted using the ImageJ software.

Intestinal biopsies were used to establish adult colonic stem cell-derived epithelial cell cultures (colonoids) that were plated as monolayers on 0.4 μm Transwell^® inserts (VWR, cat#: 76313–906), as previously described [46 (link), 52 (link)]. Briefly, colonoids from Matrigel domes were dislodged using a cell scraper and incubated in Cultrex Organoid Harvesting solution (R&D Systems, cat#: 3700-100-01) at 4°C for 90 minutes. Cells were washed, treated with TrypLE Express (Thermo Fisher Scientific, cat#: 12604021) for 1 minute at 37°C, washed and resuspended in IntestiCult^™ Organoid Growth Medium (OGM; STEMCELL Technologies, cat#: 06010) supplemented with 50 μg/mL gentamicin (Thermo Fisher Scientific) and 10 μM Rho kinase inhibitor Y-27632 (STEMCELL Technologies, cat#: 72304). Transwells^® were pre-treated with 34 μg/mL human collagen IV (Sigma-Aldrich, cat#: C5533) and cells from 1.2 to 1.4 domes were seeded per Transwell^®. Cells were incubated at 37°C (v/v 5% CO₂) and media were changed every 2 days. Growth was monitored by light microscopy for confluence and transepithelial electrical resistance (TEER) was measured with a Millicell^® ERS-2 Volt-Ohm meter, as previously described [46 (link)].