The cells were then re-suspended in 1?mL of PBS, and two 100-L samples were taken from each conical tube to be counted with the NucleoCounter. one fully enclosed bioreactor. (DH5; Life Technologies, Ontario, Canada) then grown in Luria-Broth supplemented with 30?g/mL kanamycin as previous described;35 transfection-grade plasmid DNAs were then purified from the transformed bacteria using the PureLink HiPure Plasmid Midiprep Kit, according to manufacturers protocol, with the modifications that all reagents were pre-chilled on ice prior to performing all subsequent procedures at 4C. Cells were transfected with either XtremeGENE HP DNA Transfection Reagent (Sigma), TransIT-LT1, TransIT-2020, TransIT-X2, TransIT-3D (Mirus Bio), or JetPrime (Polypus) according to manufacturers recommended protocol. In brief, plasmid DNA was diluted in OPTI-MEM (Gibco) at a concentration of 10?g/mL, then transfection reagent was added at a specified volume-to-weight ratio (v/w) in drop-wise fashion, vortexed immediately, incubated at room temperature for 16?min, then diluted in basic media with 10% FBS to a final plasmid DNA concentration of Namitecan 1 1?g/mL. Cells were then incubated overnight, up to 24?hr, at which point reporter gene expression could be observed or quantitated by either epi-fluorescent microscope or by flow cytometry. Analysis of Transfection Efficiency by Flow Cytometry Transfection efficiency was assayed by fluorescence-activated cell sorting (FACS) as previously described.36 In brief, cells were washed 3 with CMF-dPBS for 5?min, then detached from culture substrate using with 1 TrypLE Express (Gibco, Gaithersburg, MD, USA) and subsequently dissociated into single-cell suspension to be fixed in 3.7% formaldehyde in CMF-dPBS. To process transfected cells on microcarriers for flow cytometry, an aliquot of the microcarrier culture was drawn out from the bioreactor and transferred to a clean tube. Cells were dissociated from the microcarrier as per above for static culture, except 0.25% Trypsin-EDTA was used instead; dissociated single-cell suspension were subsequently passed through a 40? m cell strain prior to analysis. Samples were subjected to FACS using an Attune Acoustic Focusing Cytometer (Thermo Fischer Scientific) equipped with a 488?nm and 637?nm laser and analyzed on the Attune Software (v2.1.0). A minimum of 5,000 events were collected per sample. Analysis of intact viable cells was performed by gating the appropriate area and width of side and forward scatter to avoid cellular debris; transfection efficiency analysis was then performed by gating the fluorescent intensity of the cell population in the BL1 channel (excitation [ex] 488?nm/emission [em] 525?nm) such that the negative control (i.e., cells transfected with blank expression plasmid gWIZ) had 1%C2% autofluorescent cells. Microcarrier Preparation for Bioreactor Culture Methods Namitecan for culturing cells on microcarriers in stirred-suspension bioreactors was carried out as described previously.37 In brief, Cytodex 3 microcarriers (GE Healthcare Life Sciences) were used for all bioreactor experiments. Before the microcarriers were seeded into the 100?mL stirred-suspension bioreactors (Corning), they were hydrated, washed, and autoclaved. The desired amount of microcarriers were weighed and added to a siliconized 125?mL Erlenmeyer flask with 100?mL of Ca+/Mg+ free PBS (Life Technologies) containing 1% Antibiotic-Antimycotic Namitecan (Anti-Anti, Life Technologies). Each bioreactor was inoculated with 2 g/L of microcarriers. Three drops of Acta2 Tween 80 (United States Chemical Corporation) was added into the flask to lower the surface tension and prevent the microcarriers from sitting at the top of the liquid. The microcarriers were left to hydrate at room temperature for a minimum of 6?hr. After hydrating, 80?mL of the PBS solution was aspirated out with a 25-mL pipette, leaving 20?mL of the PBS solution in the flask with the microcarriers. Next, 25?mL of fresh PBS with 1% Anti-Anti was added to the flask. The microcarriers were settled for 5?min, then 25?mL of the PBS solution was aspirated out and discarded. This washing procedure was repeated three times. During the final washing step, 30?mL of PBS was added to the Erlenmeyer flask, resulting in a total volume of 50?mL. The Erlenmeyer flask was then sealed with parafilm and placed in a 4C fridge overnight. Before inoculation, the microcarriers were autoclaved using a liquid cycle. The PBS solution was then removed and DMEM was added to the microcarriers using a 10-mL pipette. For each bioreactor being inoculated, 20?mL of DMEM was added. The microcarriers were then split into 50-mL conical tubes (FroggaBio). Each conical tube was used to inoculate one bioreactor. The microcarriers were settled in the conical tubes, and the DMEM was aspirated out. Finally, the microcarriers were seeded into siliconized bioreactors with 60?mL of medium each. The bioreactors were placed in the incubator on.