Celldrop

Human embryonic kidney cells (HEK-293T, ATCC: CRL-11268) were cultivated in Dulbecco's modified Eagle's medium (DMEM; 10566016, Thermo Fischer) supplemented with 10% v/v fetal bovine serum (FBS; F7524, Sigma Aldrich) under a humidified atmosphere containing 5% CO₂ at 37°C. Routine sub-culture was performed every 2–3 days when cell plates were around 80% confluent. Cells were detached using 0.05% trypsin–EDTA (25300054, Life Technologies) and re-seeded in fresh medium at the desired cell density. Cell number and viability were quantified using a CellDrop automated cell counter (DeNovix).

CaCo-2
cells were cultured from liquid N₂ frozen stocks and rapidly
thawed to RT using a water bath at 37 °C; cryopreservation media
was removed with a micropipette after cells were pelleted via centrifugation
for 5 min at 125 g. Cells were resuspended in 1 mL of room temperature
Dulbecco’s modified Eagle medium (DMEM) and cultured in 14
mL of DMEM (total volume of 15 mL) with a seeding density of 3.6 ×
10⁴ cells/cm² (CaCo-2) in a T-75 cm² culture flask and left to grow in an incubator at 37 °C and
5% CO₂. Cells were subcultured at 90% confluency, and subculturing
occurred in a minimum of five times before use in uptake assays. When
cultures reached 90% confluency, media was removed and 3 mL of trypsin/EDTA
was added; the trypsinized culture flask was placed back in the incubator
for ∼10 min to detach cells. Once detached, cells were confirmed
under a microscope, the culture flask was rinsed with 6 mL of fresh
media to neutralize the trypsin/EDTA, and cells for uptake assays
were then counted utilizing a DeNovix CellDrop. Once counted, cells
were pelleted down via centrifugation at 125 g for 10 min, and the
supernatant was removed via pipetting. Cells were resuspended in 5
mL of room temperature magnesium assay buffer for cellular uptake
assay.

Melanoma cells were seeded on 6-well plates coated with Hydrogels bound with type I collagen from bovine skin or rat tail at different elastic modulus (kPA) (Softwell, Cell Guidance Systems, Cambridge, UK). After 48 h from seeding or from transfection, images were acquired by using the light channel of Bio-Rad ZOE fluorescent cell imager, cells were detached, stained with Trypan Blue solution and both viable cells excluding the dye, and nonviable cells absorbing the dye and appearing blue were counted by CellDrop automated cell counter (DeNovix, Wilmington, DE, USA).

Cell proliferation rates were examined by growing cells in a well of a six-well plate (TPP, #92006). On Day 0, 2×10³ cells were seeded in each well, and 2 ml media was provided. Presumably, because the starting cell density was low, cell growth became clearly detectable only on day 5–6. This condition allowed us to confirm the growth recovery of clone 19 on day 7–9 after cells were treated with doxycycline (1 μg/ml) and 4-OHT (0.5 μM). On the day of the measurement, the media in the well was removed, and the cells were rinsed by modified Hanks' balanced salt solution (Sigma-Aldrich, #H6648). Cells were trypsinised by 500 µl of 0.25% Trypsin-EDTA solution (Merck, #T4049) and then mixed with 500 µl of the media. 40 µl of the cell suspension was mixed with 0.4% trypan blue solution (Sigma-Aldrich T8154). The stained cells were counted using CellDrop (DeNovix).

The BAL cells were pelleted by centrifugation at 400 g for 5 minutes, then RBCs were lysed. The cells were resuspended in PBS and cell counts were quantified using an automated cell counter (DeNovix CellDrop^™). For flow cytometry, the cells were pelleted in v-shaped-bottom 96-well plates. The panel of BioLegend antibodies used for flow analysis was as follows, used at 1: 400: FITC anti-mouse MERTK (Mer) antibody (clone: 2B10C42; BioLegend); Alexa Fluor^® 700 anti-mouse major histocompatibility complex class II (MHCII; I-A/I-E) antibody (clone: M5/114.15.2; BioLegend); allophycocyanin (APC)/cyanine 7 (Cy7) anti-mouse CD11b antibody (clone: M1/70, BioLegend); Brilliant Violet 510^™ anti-mouse Ly-6G antibody (clone 1A8; BioLegend); Zombie Aqua^™ Fixable Viability Kit, Brilliant Violet 605^™ anti-mouse CD11c antibody (clone: N418; BioLegend); Brilliant Violet 650^™ anti-mouse F4/80 antibody (clone: BM8; Biolegend); phycoerythrin (PE)-CF594 rat anti-mouse Siglec-F (clone: E50–2440; BD Biosciences); and PE/Cy7 anti-mouse Ly-6C antibody (clone: Hk1.4; BioLegend). The samples were acquired using the NovoCyte Quanteon flow cytometer (Agilent Technologies) and flow data were analyzed using FlowJo^™ version 10.