Cell counter

For a viability assay by imaging, LLC1 spheroids were washed three times with 1x PBS and incubated with 2 μM Calcein-AM and 0.5 μM ethidium homodimer in 1x PBS for 10 min in the dark. Images were captured using an inverted microscope. For a viability assay via trypan blue exclusion, 20 LLC1 spheroids were collected, washed with 1x PBS, and incubated with Accutase for 5 min. Then we further dissociated the cell clumps by pipetting about 40 times using a low-binding tip to prevent cell loss. Viability was obtained by a cell counter (Countess).

GMSCs were seeded in 6-well (1 × 10⁵ cells/well) plates in growth medium with 10% FBS and no antibiotics. Cells were left untreated or incubated with F. nucleatum at multiplicities of infection (MOIs) of 10, 50, and 100 (F. nucleatum:cell ratio of 10:1, 50:1, 100:1) from 1 to 7 d, and cells were fed fresh medium every day. The cell morphology was detected by crystal violet (Solarbio) staining. Cells were fixed in 4% paraformaldehyde for 30 min and stained with crystal violet (Solarbio) for 30 min. The cells were observed immediately under the microscope and photographed (Olympus). Cell proliferation was detected by counting the cell numbers through an automated cell counter (Countstar, Shanghai, China). Briefly, cells infected with or without F. nucleatum were trypsinized, collected in a 1.5 ml microcentrifuge tube and centrifuged for 5 min at 1,000 rpm. After being washed twice with PBS, cells were resuspended in 1 ml of PBS and mixed thoroughly. Then, 20 μl of cell resuspension solution was added to the counting chamber and detected by a cell counter (Countstar). Cell proliferation rate was detected by the 5-ethynyl-20-deoxyuridine (EdU) labeling assay according to the instructions of an EdU Apollo DNA in vitro kit (RIBOBIO, Guangzhou, China).

Cell viability in the 2D and 3D cell culture systems was determined by trypan blue staining. The 2D-MSCs and 3D-MSCs were suspended and stained with a trypan blue solution (Solarbio, Beijing, China). Then, the number and viability of living cells were counted by the cell counter (Countstar, Shanghai, China).

Confluent cells were trypsinised and neutralised by adding DMEM supplemented with 10% FCS and Penicillin and streptomycin. Centrifugation was done at 1800 rpm for 5 min at 25 °C and cells were resuspended in DMEM. The Countess Cell Counter was used for cell counting to give a final cell density of 5000 cells per microliter. Cellular foci of 4 µL containing a total of 20,000 cells were added to plastic dishes or to the ECMs. To prevent cell death due to evaporation of media, an extra 100 µL of DMEM media was added to the cellular foci and incubation continued for 2 h. A further 3 mL of DMEM with 10% FCS was then added and incubation continued for 24 h. In a separate experiment, cells were incubated with anti-α2 integrin blocking antibody for 30 min before plating. Images were taken at 0 h and at 24 h with a microscope. Image J (version 1.52g, National Institutes of Health; Bethesda, MD, USA) was then used to measure the area of migrating mass of cells. Migration on ECMs was normalised to that on plastic dishes.

Chemotaxis of fmDCs or cryoim-mDCs was assessed by their migration through an 8 μm polycarbonate membrane (Corning, NY, USA) using CCL19 as the chemoattractant. DCs were seeded (5 × 10⁴ cells in 100 μL of RPMI 1640) in the upper chamber in 24-well cell culture plates and the lower chamber contained 500 μL of RPMI 1640 with or without CCL19 (final concentration, 500 ng/mL). Each condition was set up in three repeats. The plates were incubated for 4 h at 37 °C in 5% CO₂. The cells remaining in the upper chamber were removed and the migrated cells were harvested by washing the bottom side of the inserts and the wells 3 times. The total migrated cells were counted by use of Cell Counter (IC1000, Countstar, Shanghai, China) and the results were expressed as the mean number of migrating cells ± standard deviation (SD).

Cauda epididymides from 6-month-old mice were removed and cut in 500 μl of DMEM. The medium was incubated at 37 °C for 7 min to release sperm. After diluting them 50 times, the sperm were analyzed with a cell counter (Countstar).