Cloneselect imager

Cells in each group were seeded into 96‐well plates (5 × 10³ cells/well) and cultured overnight. Then, the corresponding reagents were added. After 24, 48, and 72 hours, cell proliferation was quantified by cell confluence with a CloneSelect Imager (Molecular Devices, Sunnyvale, CA).

24-well plates were seeded at 10 000 cells per well in 500 μL medium. They were imaged daily on the CSI (CloneSelect Imager, Molecular Devices) which measured the confluence (representing cell number) of the wells, to generate a growth curve. 200 μL medium was added to each well every three days. Plates were incubated at 37°C, 5% CO₂ in a loosely sealed box containing damp paper towels to reduce the effects of evaporation in the outside wells.
In a 96-well plate cells were seeded at a density of 5000 cells in 120 μL media per well. After 72 hours, Alamar blue reagent (Invitrogen) was added to the wells in a 1:10 dilution. Fluorescence was then measured every hour for 3 hours using a SPECTRAmax, GEMINI XPS fluorescence reader (Molecular Devices).

The wound healing (scratch) assay was performed as described elsewhere [39 (link)] with some changes. In brief, cells were seeded in 96-well cell culture plates (6 × 10⁴ cells/well) and grown for 24 h. After 8 h, the wells were scratched with a sterile 10 μL pipette tip. Then, the cells were washed with PBS and incubated with EVs for additional 24 h (for mel P cells) or 48 h (for Het-1A cells). Pictures were analyzed after 0, 24, and 48 h at 20× magnification at CloneSelect Imager (Molecular Devices). The center of the plate was marked as the central reference point to ensure recording of the same area during the time course. Digital images were taken, and the scratch area was quantified using the ImageJ (NIH, Bethesda, MD, USA) and MS Excel (Microsoft, Redmond, WA, USA) software by the percentage measurement of the scratch surface occupied by the migrating cells. In each experiment, the duplicate measurements were averaged.
For investigation of the Het-1A cells invasion, the Abcam migration/chemotaxis assay kit (ab235694, Abcam, Cambridge, UK) based on cell migration through the 8 µm pored membrane was used. Cells were seeded in the migration chambers in 24-well plates (2 × 10⁵ cells per well), incubated with the normal or acidic EVs for 48 h, and quantified according to the manufacturer’s protocol.

The effect of RRS1 knockdown on colony formation ability of the TPC1 cells was detect by plate colony formation assay. Forty-eight hours after short hairpin RNA affection, cells were plated at limiting dilution (800 cell per well) in 6-well format in triplicate. Cells were fed twice weekly until control colonies began to merge. Samples were fixed with 4% formaldehyde and stained with Geimsa. Plates were scans for the number of colonies under CloneSelect Imager (Molecular Devices).

Cell lines for the VEGFR-IgG fusion protein were generated using suspension-adapted CHO-k1 cells in chemically defined medium. In addition, 2 × 10⁶ cells were transfected by electroporation with 20 μg of the expression vector using a proprietary recombination-based transfection protocol. Transfected cells were monitored daily and subject to media change and antibiotic selection with puromycin dihydrochloride (10 μg/mL) every 48 hrs for 10–15 days. Stably transfected cells were selected for high-expressing clones with MoFlo-XDP (Beckman Coulter, Brea, CA, USA). Growth and specific productivity rates (SPR) were monitored by Clone Select Imager (CSI, Molecular Devices, San Jose, CA, USA) and by ELISA assay according to the protocol in Brezinksy et al. [30 (link)]. The final top three high-expressing clones were cryopreserved and tested under fed-batch conditions.