Cellometer auto 2000 cell viability counter

The sex of young mice was determined using anogenital distance and the presence of inguinal mammary teats [69 (link)]. Male and female cells were prepared separately. Cerebellar granule cell cultures were prepared as described [70 (link)]. Briefly, cerebella were incubated in 0.04% of trypsin, 4% of DNase I, and 0.8 mM of MgCl₂ in HBSS for 15 min at 37 °C and mechanically dissociated. Dissociated cells were mixed with ViaStain™ AOPI Staining Solution, prepared, and counted according to Cellometer Auto 2000 Cell Viability Counter (Nexcelom) manufacture information. Subsequently, dissociated cells were seeded on 48-well plates coated with 0.01% PLL.

CCK-8 assay and cell count assay were used to determine cell proliferation. For CCK8 assay, 3 × 10³ cells were seeded into the 96-well plates and incubated for several days in a row. 10 μl CCK-8 solution (Beyotime, catalog number: C0043, China) was added to each well at the same time pointed each day, and cells were incubated for 2 h. Then, the optical density (OD) values in the wavelength of 450 nm were determined to assess cell viability using the microplate reader Varioskan FLASH (Thermo Fisher Scientific, USA). For cell count assay, 1.2 × 10⁴ cells were seeded into the 24-well plates and counted on day 2 and day 4 after seeding using cellometer auto 2000 cell viability counter (Nexcelom, USA).

Bone marrow was collected aseptically from the sternebrae of horses being euthanized for unrelated reasons immediately following euthanasia. Using an 11-gauge Jamshidi bone marrow biopsy needle (VWR Scientific, Bridgeport, NJ) and 60 mL syringe containing 30,000 U of heparin, 30 mL of bone marrow was aspirated. Bone marrow samples were processed via density centrifugation with Ficoll-Paque Plus (GE Healthcare, Chicago, IL, USA) prior to seeding into flasks containing medium consisting of Dulbecco's Modified Eagle Medium (DMEM) with 1 g/L of D-glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate (ThermoFisher Scientific, Hampton, NH), penicillin (100 U/mL)-streptomycin (100 μg/mL) solution (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS) (VWR Life Science Seradigm, VWR, Radnor, PA), and basic fibroblastic growth factor (bFGF, 1 ng/mL) (Invitrogen, Carlsbad, CA). Medium was changed every 48 h. Cells were passaged when they reached ~80% confluency using Trypsin-EDTA Cell Dissociation Reagent (ThermoFisher Scientific, Waltham, MA). Passage 2 (P2) cells were used for differentiation assays. Cell number and viability was determined using the Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience, Lawrence, MA) and ViaStain™ AOPI staining solution (Nexcelom Bioscience LLC, Lawrence, MA).

Cells were resuspended in RPMI media to obtain a single-cell suspension with high cell viability. Next, cells were stained with a live/death dye (DAPI) and dead cells were removed using fluorescence-activated cell sorting (FACS). Live cells were resuspended in PBS buffer and recounted using AOPI staining and the Nexcelom Cellometer Auto 2000 Cell Viability Counter. Finally, cells from four independent biological replicates were pooled in equal cell numbers into a single-cell suspension for each condition. Cell suspensions were processed for single-cell RNA-sequencing using the 10×-Genomics 3′ v2 kit^{33 (link)}, as specified by the manufacturer’s instructions. Namely, 1 × 10⁴ cells from each condition were loaded in separate inlets of a 10×-Genomics Chromium controller in order to create GEM emulsions. The targeted recovery was 3000 cells per condition. Emulsions were used to perform reverse transcription, cDNA amplification and RNA-sequencing library preparation. Libraries were sequenced on the Illumina HiSeq 4000 platform, using 75 bp paired-end reads and loading one sample per sequencing lane.

Mice were sacrificed, the trachea exposed and a small incision was made to insert a blunted cannula. The lung was flushed three times using 0.8 ml PBS each time. The bronchoalveolar lavage fluid (BALF) containing the infiltrating cells was collected, viable cells were counted using the Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience) and analyzed by flow cytometry.

Cells were seeded in T25
tissue culture flasks and incubated for approximately 24 h before
Au³⁺ biomineralization treatments with chloroauric acid
at final concentrations of 0.00, 0.10, or 0.20 mM Au. Cells were incubated
with the Au³⁺ ions overnight followed by cell collection
via trypsinization. Finally, cells were resuspended in PBS and briefly
admixed with an Nexcelom AO/PI dye assay kit at a 1:1 (v/v) ratio
immediately before 20 μL was dispensed to Cellometer Cell Counting
Chambers for quantitation of viability using a Cellometer Auto 2000
Cell Viability Counter (Nexcelom) per the manufacturer’s instructions.^{99 (link)}