Hemocytometer

Normal C57BL/6 female mice (6–8 weeks old) were procured from the Jackson Laboratory (Bar Harbor, ME) and used when mice were about 12 weeks old. Animals were housed at the University of South Carolina Animal facility. All necessary care and maintenance of the animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals.
Spleens from mice were collected and placed in complete RPMI 1640 medium. Immediately, single-cell suspensions of splenocytes were prepared and RBC lysed as described previously.^{49 (link)} Cell viability was determined on a hemocytometer (Hausser Scientific, Horsham, PA) by trypan blue dye staining of the cells and using an inverted phase-contrast microscope (Nikon, Inc., Melville, NY).

HEK293T cells and Preadipocytes were stained with CellBrite™ Green (#30021) and Red (#30023) Cytoplasmic Membrane Labeling Kits respectively using manufacturer’s protocol. Briefly, cells were suspended at a density of 1,000,000 cells/mL in their respective normal growth medium. 5 μL or 10 μL of the Cell Labeling Solution was then added per 1 mL of cell suspension for HEKs and Preadipocytes respectively. Cells were then incubated for 20 minutes (HEKs) or 40–60 minutes (Preadipocytes) in a humidified incubator containing 5% vol/vol CO₂. Cells were then pelleted by centrifugation at 1,200 rpm for 4 min. After centrifugation, the supernatant was removed, and cells were washed in warm (37 °C) medium. Cells were centrifuged again, and the process was repeated for a total of 3 growth medium washes for HEKs and 1–3 growth medium washes for Preadipocytes. Cells were then centrifuged a final time at 1,200 rpm for 4 minutes and resuspended in ice-cold PBS (Corning, 21–040-CV) for a final concentration of 700 cells/μL adjusted using a hemocytometer (Hausser Scientific). The cells were then stored on ice throughout the μCB-seq device operation.

The concentration of beads in the sample before and after use was determined using a hemocytometer (Cat#:1492, Hausser Scientific). A 10 μl of sample solution was injected in the hemocytometer without dilution and imaged using a florescence microscope (IX81, Olympus). The average bead count in four cells of the hemocytometer, identified by overlaying phase and green florescence images, was used to calculate final concentration of beads per microliter.