Countess automatic cell counter

For migration assay, the cells were incubated for 48 h post‐transfection and then, seeded on the membrane surface of the upper chamber (24‐well, Corning, Corning, NY, USA) coated without Matrigel (BD Biosciences), whereas the lower chamber (Corning) was added with DMEM with 10% FBS to undertake the nutritional attractant. For the invasion assay, the whole process was similar to migration assay except that the membrane of upper chamber was added with Matrigel. After 48 h, the cells which had migrated or invaded into the bottom surface of the membrane were dyed with crystal violet (0.5% w/v) for 30 min after the fixation with methanol. The invaded or migrated cells were counted using a Countess automatic cell counter (Invitrogen) in five randomly chosen fields under inverted microscope (Nikon TE‐300).

MOLT-4 and CCRF-CEM cells were obtained from ATCC (CRL-1582 and CCL-119) and grown by standard methods in RPMI 1640, with 2 mM GlutaMax and 10 % FBS (Invitrogen; 61870-010, 10108-57), in a humidified incubator at 37 °C under 5 % CO₂. At least 3 days before experiments were conducted, cells were introduced to serum-free medium (Advanced RPMI 1640, containing 2 mM GlutaMax; Invitrogen; 12633-012, 35050-038). The medium was refreshed the day before starting the experiment. For experiments, cells were centrifuged at 201×g for 5 min and resuspended in serum-free medium containing DMSO (0.67 %) at a cell concentration of 5 × 10⁵ cells/mL. The cell suspension was seeded in triplicate, with 1 or 2 mL applied to a 24-well or 12-well plate, respectively. At the indicated times, the cells were removed by centrifugation, and the spent medium was frozen at −80 °C. Cell number, size, and viability (Trypan blue exclusion) were determined by counting cells on a Countess automatic cell counter (Invitrogen).

Detached astroglial cells were reseeded in 24-multiwell plates (12,000 cells/well) with 5% FBS. Three days later these cells were exposed to ammonia (1, 3 or 5 mM NH₄Cl) for 1, 3 or 10 days. Both control and treated cells were then washed with PBS, detached with trypsin, washed again, and centrifuged (100 g for 5 min) in culture medium. After suspension in PBS, the cells were stained with trypan blue to identify those alive and dead; enumerating was performed using a Countess automatic cell counter (Invitrogen) and cell counting chamber slides. The experiment was performed in duplicate with six wells used for each duration and ammonia concentration.

Anterior limbal stromal tissues obtained from six donor corneas were trimmed to tiny pieces, pooled together, and then separated into three portions. Each portion was digested using a different collagenase type (1 mg/ml): (i) collagenase A (research-grade, Roche #11,088,793,001), (ii) Celase (GMP, Worthington #1235–01), or (iii) NB6 (GMP, Nordmark #N0002779), at 37 °C for 6 h (Table 1). After the digest was filtered through a 40-μm cell strainer, followed by washes and centrifugations, the resulting cell pellet was resuspended in sterile PBS (2 ml volume), loaded to a Ficoll-Paque gradient (4 ml volume), and centrifuged at 1,000 g for 20 min. This step removed tissue debris, and the cell enriched fraction in the interface layer was harvested. After PBS washes, the cells were resuspended in JM-H medium (0.5 ml). Viability count was performed by 0.4% trypan blue dye exclusion using a Countess Automatic Cell Counter (Invitrogen). Each sample was counted three times, and the mean ± standard deviation (SD) was calculated.

Human primary monocytes were cultured in CMs or ordinary medium (serum‐free X‐Vivo 15) with/without control additives (rS100A4, M‐CSF, or GM‐CSF) for 7 days. Cultures were observed by microscopy to evaluate morphological changes (images were obtained using an Olympus IX81 inverted microscope and the Olympus cellp imaging software, Tokyo, Japan). Cells harvested by EDTA and gentle scraping were analyzed for the levels of polarization markers by flow cytometry.
THP1 monocytes (25 000 cells·cm⁻²) were cultured in CMs or ordinary medium RPMI (all supplemented with 10% FBS) with/without rS100A4 for 7 days. Immature, unattached viable (i.e., trypan blue‐negative) monocytes were counted by a Countess automatic cell counter (Invitrogen, Carlsbad, CA, USA). The harvested cells were used for analysis of gene expression.
For analysis of cytokines, the THP1 cells were cultured in CM‐S100A4 or CM‐Ctr for 7 days and washed, and the same number of cells from both conditions were cultured further in serum‐free ordinary medium (0.08 mL·cm⁻²) for 3 days, before their growth medium was analyzed.

Trypan-blue (EBT-001 EnTek, Lebanon, OR, USA) was mixed in a 1:1 ratio with the cells. Then, the cells were counted using a countess automatic cell counter (Invitrogen, Waltham, MA, USA). Live cells were unstained, and dead cells assimilated the dye.