Luna fl automated cell counter

Cells in mid-log phase were diluted to 5,000 / ml and supplemented with 10 μg/ml E. pacifica bacteria (diluted 1:1000 from a stock of 10 mg/ml in AKSW). 500 μl of culture was aliquoted into each well of a 24-well plate (Fisher Scientific Cat. No. 09–761-146) and cultured at 22°C. Plates were kept in a Tupperware box with dampened paper towels and the lid loosely affixed to prevent cultures from drying out but to allow gas exchange.
Every 12 hours for 96 hours, 3 wells from each strain were fixed with 10 μl of 16% paraformaldehyde (Fisher Scientific Cat. No. 50–980-487) and stored at 4°C. After all time points were collected, each sample was counted by vortexing the sample at high speed for 10 seconds to fully mix the sample, then aliquoting 10 μl into a counting slide (Logos Biosystems Cat. No. L12001 [disposable] or L12011 [reusable]) and counting using a Luna-FL automated cell counter (Logos Biosystems, Anyang, KOR; Cat. No. L20001).

The human neuroblastoma cell line (SH‐SY5Y) was used in this investigation. SH‐SY5Y cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Cat. No. 11995065; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin–streptomycin (Thermo Fisher Scientific). Cells were incubated in a Falcon 25‐cm² Rectangular Canted Neck Cell Culture Flask with Vented Cap (Cat. No. 353108; Corning, Corning, NY, USA) or a Falcon four‐well Culture Slide (Cat. No. 354114; Corning) and maintained at 37°C in a humidified incubator under 5% CO₂ atmosphere. Cell counting was verified by the trypan blue dye exclusion method using a LUNA‐FL automated cell counter (Logos Biosystems, Gyeonggi‐do, South Korea) and dedicated using LUNA Cell Counting Slides (Logos Biosystems). SH‐SY5Y cells were divided into different groups with or without several treatments as mentioned later.

Approximately 2.5 × 10⁵ cells were plated in to each well of a 6-well plate. At 24, 48, and 72 h after plating, cells were lifted with 0.05% Trypsin and counted via hemocytometer and/or a LUNA-FL automated cell counter (Logos Biosystems, Anyang, Kyonggi-do, South Korea). Media were refreshed on remaining cells every 24 h to avoid nutrient/additive deficiency during the time frame of the experiment and to allow for constant proliferation.

Spleens were harvested from mice, placed in RPMI 1640 (Mediatech, Manassas, VA), and diced with frozen slides in Hanks balanced salt solution medium (Cellgro, Manassas, VA) to produce cell suspensions. Red blood cell lysis was performed using 2 ml/spleen of 1X ACK lysing buffer (Life Technologies). Cells were then washed in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (Atlanta Biotech) and 0.09% sodium azide (Sigma-Aldrich, St. Louis, MO), followed by passage through 70-μm-pore-size and 40-μm-pore-size nylon filters (BD Falcon, Bedford, MA), and the cells were counted using a Luna-FL automated cell counter (Logos Biosystems, Gyunggi-Do, South Korea).

Cultured keratinocytes were harvested and resuspended in ice-cold buffer containing 0.5% FCS in DPBS. A small aliquot of the cell suspension was used to determine the percentage of live/dead cells by stained with Acridine Orange/Propidium Iodide (AO/PI) Cell Viability Kit (Vita Scientific, Beltsville, MD, USA, #LGBD10012) and counted by using LUNA-FL automated cell counter (Logos Biosystems, Annandale, VA, USA). Approximately 3000 live cells from each group were loaded into one channel of the Chromium Chip G using the Single Cell reagent kit v3.1 (10X Genomics, Pleasanton, CA, USA) for capturing single-cell Gel Bead Emulsion using the Chromium controller. Following single-cell capture, cells were lysed, and cDNA of individual cells were synthesized within the Gel Bead Emulsion and were used for library preparation. cDNA was amplified and size-selected using SPRISelect (Beckman Coulter, Krefeld, Germany, #B23317) following the manufacturer’s protocol. In all, 25 ng of the size-selected cDNA of each sample was used to construct Illumina sequencing libraries. Libraries were pooled at equal concentration and sequenced on the NovaSeq SP100 or NextSeq2000 Illumina sequencing platform at 50,000 reads per cell.

Larvae of G. mellonella were purchased from waxworms.net (St. Marys, OH, United States) and infection assays were performed similar to previous reports (Cotter et al., 2000 (link); Ames et al., 2017 (link)). Upon arrival, larvae were held at room temperature in the dark, without food for 24 h before injection. C. albicans strains were grown to saturation in YPD and cells washed three times with PBS, sonicated for 10 s, and counted using a Luna-FL automated cell counter (Logos Biosystems). Larvae (n ≥ 40 per strain) of similar size and color were injected with 6 μl of 2 × 10⁵ cells into their lower left proleg using a Hamilton syringe. An additional group was injected with PBS to serve as a negative control. Injected larvae were incubated in petri dishes at 37°C in the dark for 5 days. Death was measured every 24 h based on the ability of the larvae to flip over after being placed on their backs. Survival data were plotted using the Kaplan–Meier survival curve and statistical analysis was performed using a log-rank Mantel–Cox test.

Single-cell suspensions of endolymphatic sac epithelia were prepared from E12.5, E16.5, P5 or P30 C57BL/6J mice of both sexes for RNA-seq or qPCR analysis. Inner ears were removed from four to five mice and placed in ice-cold DMEM/F12 (GIBCO). The endolymphatic sac was harvested together with surrounding connective and osseous tissues. The tissues were pooled and incubated in 1 mg/ml collagenase (LS004194, Worthington, Lakewood, NJ) and 1 mg/ml dispase (LS02100, Worthington) in DMEM/F12 at 37°C for 10 min. The tissues were then transferred into 10% fetal bovine serum (FBS) (GIBCO) in DMEM/F12 where the epithelia were isolated from surrounding tissues. The epithelia were transferred to 50 µl of 0.125% trypsin/EDTA (GIBCO) in phosphate-buffered saline (PBS) in a 1.5-mL tube and incubated for 15 min at 37°C. Then 50 µL of 10% FBS in DMEM/F12 was added to the tube to stop digestion. The epithelia were gently triturated with a 200-µl micropipette tip to complete the dissociation. Cell concentration was measured using Luna-FL automated cell counter (Logos Biosystems, Annandale, VA) and adjusted to 250,000 to 300,000 cells/ml by adding 5% FBS in DMEM/F12. To assess cell viability after cell capturing, the cells were labeled with 1:1000 of calcein-AM or ethidium homodimer-1 (EthD-1) (LIVE/DEAD cell viability assay, Thermo Fisher Scientific, Waltham, MA) and placed on ice until capture.