Enzyme free cell dissociation solution

The ADAM10 and ADAM17 moderately-specific FRET enzyme substrate PEPDAB005 (green-color fluorescence with optimal excitation and emission wavelengths of 485 nm and 530 nm, respectively) 21 (link), 22 (link) was obtained from BioZyme Inc (Apex, NC). Cell-membrane selective lipophilic fluorophore DiD (near-infrared fluorescence with optimal excitation and emission of 644 nm and 665 nm, respectively), was purchased from ThermoFisher Scientific (Pittsburgh, PA) as a Vybrant cell-labeling solution. Cell-nuclear DNA specific Hoechst 33342 fluorescent dye (blue fluorescence with optimal excitation and emission of 350 nm and 461 nm, respectively) was purchased from ThermoFisher Scientific. Phycoerythrin (PE)-conjugated mouse monoclonal antibodies (mAb) to human ADAM10 and ADAM17 and corresponding isotype control mAb (red-color fluorescence with excitation and emission wavelengths of 564 nm and 573 nm, respectively) were obtained from R&D Systems (Minneapolis, MN). Paraformaldehyde (PFA) (10% aqueous solution) and Glutaraldehyde (GAL) (2.5% solution in 0.1 M Millonig's Sodium Phosphate Buffer, pH 7.2) were purchased from Electron Microscopy Sciences (Hatfield, PA). RPMI-1640 and DMEM cell-culture media, fetal-calf serum (FCS), Trypsin and enzyme-free cell-dissociation solution were obtained from GIBCO-Life Technologies (Grand Island, NY).

For detection of PS on PRRSV envelope, MARC-145 cells were washed with PBS and dissociated by using an enzyme-free cell dissociation solution (catalog no. 13151014; Gibco). The cells were then collected at 4°C for 1 h and inoculated with virions at a multiplicity of infection (MOI) of 10 or with PBS as an unbound control. The cells were washed three times with cold PBS, resuspended in annexin-binding buffer, and incubated with annexin V-conjugated Alexa Fluor 488 at 4°C for 20 min. After incubation, the cells were added with annexin-binding buffer, mixed gently, and kept on ice. PS detection was preceded with FCM immediately.
Purified virions in PBS were spotted onto nitrocellulose membranes (Pierce, Rockford, IL), and PBS was spotted onto membranes as a negative control. Membranes were dried and blocked with 5% bovine serum albumin (BSA) in PBS at 4°C overnight. Next, the membranes were incubated with the anti-PS MAb or anti-PRRSV GP5 MAb as a viral loading control for 1 h at 37°C. After three washes with PBS plus Tween 20 (PBST), the membranes were incubated with HRP-conjugated goat anti-mouse IgG antibody and detected by enhanced chemiluminescence (ECL) Plus reagent (Solarbio, Beijing, China).

mESCs were cultured according to a modified
protocol based on previously reported methods
from Shen and Qu (37 (link)). Briefly, gelatin (0.1%) in phosphate-buffered saline (PBS) was poured into
96-well culture plates. The plates were incubated
for 30 minutes at room temperature. The excess
gelatin solution was removed by aspiration and the
plates were allowed to air dry for 20 minutes at
room temperature.
mESCs were suspended in 25 cm² gelatin-coated
TCP at a density of 1-3×10⁵ cells/10 ml in DMEMKO
with 20% (v/v) heat-inactivated FBS and essential
factors [100 U/ml penicillin, 100 mg/ml
streptomycin, 2-ME (2 mM), NEAA (0.1 mM),
L-Glu (2 mM) and 10 ng/ml LIF]. Cells were incubated
under conditions described above with a
change of media every 24 hours until the mESCs
were approximately 70% confluent. Then, mESCs
were trypsinized (0.25% trypsin) and suspended at
a density of 2500 cells/100 μl (in 96-well plates) in
DMEM-KO and RPMI-1640 (both supplemented
with 20% FBS) with essential factors. mESCs cultured
on PCL were seeded onto PCL that had been
secured into 96-well plates (Corning Inc., USA)
at a density of 2500 cells/100 μl. mESCs grown
on TCP at the same density were used as control
cells. Cells cultured on PCL were harvested by
an enzyme-free cell dissociation solution (Gibco,
USA). After 48 and 96 hours, we performed trypan
blue cell staining and counted the viable cells.