Facscanto b

In order to provide a benchmark comparison for our acoustofluidic devices, SEM and flow cytometry were applied to analyze separation performance. For SEM imaging, separated particles were dropped on a clean silicon wafer, dried in a chemical hood, sputtered with gold, and then observed with an electron microscope (FEI XL30, FEI, USA). For flow cytometry, 50 μL of mixed E. coli and RBCs, separated E. coli, and separated RBCs were diluted with 1 mL PBS in a 5 mL tube (352235, Falcon, USA), respectively. After that, the samples were tested by a flow cytometer (BD FACSCanto B, USA) and analyzed using FlowJo software (FlowJo, FlowJo, LLC, USA).

Tumors were harvested and digested with 20 ¼g/ml type II DNase I (Sigma, D4527) and 1 mg/mL collagenase IV (Worthington LS004186) to obtain single cell suspensions. Spleen and blood samples were processed with red blood cell lysis buffer (1 mM ammonium bicarbonate and 114 mM ammonium chloride). Antibodies (1:100 ratio) and Zombie Aqua™ live/dead dye (1:500 ratio) (Biolegend, 423102) were added to cell suspensions in PBS and incubated for 20 min on ice before flow analysis on BD FACS CantoB. Data were analyzed on FlowJo and represented as percentages of positive cells. Antibodies: FITC rat anti-mouse CD45 (Biolegend, 103108), APC rat anti-mouse CD3 (Biolegend, 100235), PE rat anti-mouse CD4 (Biolegend, 116005), and PE/Cyanine7 rat anti-mouse CD8a (Biolegend, 100721).

To analyze cell viability with a fluorescence microscope, stool samples liquefied using either a standard method or our acoustofluidic device were using a commercial staining kit (Live/Dead BacLight, L7007, Invitrogen, USA). After 15 min incubation in dark at room temperature, a 2 μL drop of each liquefied stool sample was placed on an agarose pad to immobilize the bacterial cells.^{55 (link)} Then, the agarose pad was analyzed using an inverted microscope (Eclipse Ti, Nikon, Japan) equipped with a 100× oil immersion objective.
In order to analyze cell viability with a flow cytometer, the liquefied stool sample was first filtered with a 5 μm filter (7037350, Sterlitech, USA) to isolate bacterial cells and then stained with a Live/Dead BacLight kit (L34856, Invitrogen, USA). After a 15 min incubation, 10 μL of liquefied stool sample was diluted with 987 μL of PBS and then transferred to a 5 mL tube with cell-strainer cap (352235, Falcon, USA) for flow cytometry (BD FACSCanto B, USA). Escherichia coli (E. coli) bacterial cells purchased from ATCC (8793) were cultured in Miller’s LB Broth (20716002, Cellgro, USA) and used for flow cytometry condition setting.