It proves that this method can enhance the ratio of droplets encapsulated with single cells and has limited adverse effects on THP-1 cell viability when low concentrations of OptiprepTM are used. study in a non-toxic environment and is expected to broadly facilitate single-cell analysis. is the sedimentation velocity, g is the gravitational acceleration, is the particle diameter, and are the particle and fluid densities, respectively, and is the fluid viscosity. From Equation (1), we know that the sedimentation velocity will increase with effective particle diameter and this indicates that aggregation of cells results in increased sedimentation velocity. 2.2. Poisson Distribution Here, the Poisson distribution is used as an informative predictor for the rate of single-cell encapsulation when the target cells are smaller than the droplets volumetrically and are distributed homogeneously in an aqueous solution. The Poisson distribution, which is a discrete probability distribution, has been used to calculate the probability of a single cell in one droplet during encapsulation, assuming there is random dispersion of cells in the sample and constant flow velocity (shown in Table S1). The use of OptiPrep? can achieve uniform suspension of cells in the sample by tuning the aqueous density to that of cells. The probability of one droplet containing cells can be dictated by is the average number of cells per droplet, is the concentration of cells in aqueous solution with unit of cells/mL, and is the volume of each droplet. By replacing in Equations (2) with (3), the probability of droplets containing cells at different lithospermic acid droplet sizes and cell concentrations can be calculated by Poisson distribution using MATLAB (MathWorks, Natick, MA, USA). 3. Materials and Methods 3.1. Device Design and Fabrication The droplet-based microfluidic device used in this study consists of two inlets for the perfusion S1PR4 of disperse phase and continuous phase, connecting microchannels with an aspect ratio of height/width = 1:2 (height: ~40 m; width: ~80 m), a rectangular observation chamber of 2 0.65 cm, and one outlet (shown in Figure S1). The geometry we used here was T-junction, in which the oil flowed horizontally towards the observational chamber, and the aqueous lithospermic acid phase flowed vertically and sheared into uniform droplets. This droplet-based microfluidic device was fabricated using standard soft-lithography techniques, including: (i) mask design via computer-aided design software; (ii) mylar mask printing; (iii) fabrication of the SU-8 (SU-8 2035 or 2050, MicroChem, Newton, MA, USA) master mold; (iv) casting of poly(dimethyl siloxane) (PDMS) (Sylgard 184, Dow Corning, Midland, MI, USA); and (v) air plasma treatment on the surfaces of the glass substrate and PDMS slabs for irreversible covalent bonding. 3.2. Cell Culture and Preparation The acute monocytic leukemia THP-1 cell line was obtained from CellBank Australia. Cells were cultured in a vertical T-75 flask filled with 12 mL of the complete growth medium: 90% RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA), and supplemented with 1% penicillin-streptomycin (Thermo Fisher Scientific, USA); and kept in an incubator (Thermo Fisher Scientific, USA) which provides sterile conditions at 37 with 5% carbon dioxide. THP-1 cells were inoculated in fresh complete growth medium at an initial concentration of 2 105 cells/mL. The number and viability of THP-1 cells were measured by the Trypan blue-based TC-20 automated cell counter (Bio-Rad, Hercules, CA, USA). Normally, to acquire enough volume (e.g., 1 mL) of cell suspension (e.g., 6 106), two flasks of cells are cultured for four days simultaneously, then spun down and suspended with fresh medium which adjusts the cell density to the desired value to be used before the viability drops down to 95%. When cell number and viability both satisfied the requirements, THP-1 cells were used to perform encapsulation in microfluidic droplets. 3.3. Encapsulation of Single Cells in Water-in-Oil Droplets Oil phase, Novec? 7500 Engineered fluid (3M, St. Paul, MN, USA) mixed with 2% Pico-Surf? 1 (Sphere Fluidics, Cambridge, UK) as surfactant, and aqueous phase cells in culture medium and OptiPrep? (Sigma-Aldrich, USA), were delivered via two syringe pumps (PHD 2000, Harvard Apparatus, Holliston, MA, USA; Chemyx, Fusion 200, Stafford, TX, USA) into the microchip to lithospermic acid produce cell-encapsulated microdroplets. The fluorinated ethylene propylene (FEP) tubing (IDEX, Lake Forest, IL, USA), with an inner diameter of 0.5 mm, was used for connecting the syringes to the microchip inlets. The microfluidic chip was used to produce uniform cell-laden droplets of different sizes by tuning the flow rate of the oil phase and aqueous phase. 3.4. Measurement of Cell Density and Viability with the Presence of OptiPrep? The density gradient centrifugation method is considered as a golden standard to measure.