The number of viable and dead cells in suspensions was determined by staining with trypan blue (Sigma, St. Louis, MO, USA). An equal volume of the cell suspension was added to 25 µL of PBS with 0.4% trypan blue and mixed. After 2 min, the number of blue cells (dead cells) and transparent cells was counted using a Luna II cell counter (Logos Biosystems, Anyang, Republic of Korea). The percentage of live and dead cells was also calculated by a cell counter.
Luna 2 cell counter
Manufactured by Logos Biosystems
The Luna II cell counter is a compact, automated device designed for precise cell counting and viability analysis. It utilizes Logos Biosystems' proprietary fluorescence imaging technology to provide accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Trypan blue, by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 2, by R&D Systems (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Multiskan skyhigh spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Collagenase, by Fujifilm (1 mentions)
Dnase, by Roche (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Spectramax id3 microplate reader, by Molecular Devices (1 mentions)
Dead cell removal kit, by STEMCELL (1 mentions)
Anacardic acid, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Hi fbs, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
14 protocols using luna 2 cell counter
1
Viable and Dead Cell Enumeration
2
Fungal Cultivation and Metabolite Analysis
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MM agar plates were streaked with conidia from glycerol stocks or from isolated single colonies that were determined by colony PCR. These plates were incubated at 33 °C for several days till plates were fully grown. Fresh conidia suspensions were prepared by harvesting conidia from these plates with sterile 0.9% NaCl solution. The harvested conidia were counted on the LUNA II cell counter (Logos Biosystems). Non-baffled shake flasks (500 mL) were filled with 100 mL M12 ++ medium and inoculated with 1.0 × 106/mL conidia and incubated at 35 °C and 0 RPM. Flasks were weighed when empty, after inoculation and each day before sampling. Evaporation is calculated from the measured weight of the flasks and used to correct measured concentrations of organic acids and glucose by HPLC (see below). Error bars in graphs of flask cultivations indicate the standard error of the mean. All flask cultivations were performed in duplicate.
3
Quantifying 3D Spheroid Viability
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First, spheroids were dissociated to a single-cell suspension with trypsin/EDTA (Gibco, cat. no. 25200072, Waltham, MA USA). Then, 100 µl of trypsin/EDTA Spheroids was incubated for 10–20 min at 37 °C and pipetted several times to separate the cells. Then, the cells were centrifuged at 300g for 3 min, the supernatant was removed, and 100 µl of medium (Gibco, cat. no. 10010056, Waltham, MA, USA) was added to the cells and pipetted several times to separate the cells completely. In the final step, 10 µl of cells was added to 10 µl trypan blue and counted (the Luna-II™ cell counter, Logos Biosystems, Aligned Genetics, Inc). The workflow is shown in Fig. 2 . Each viability test was performed on 4 different samples in each measurement under the same conditions.![]()
Workflow for investigation of spheroid viability after harvesting 3D spheroids from 5D microplates.
4
Single-Cell RNA-Sequencing of Suspended Cells
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Suspended cells (MC, MCEC, HCAEC) were delivered to the UB Genomics and Bioinformatics Core (UBGBC) for cell counting on the Logos Biosystems LUNA II cell counter using 0.4% Trypan Blue for cell viability. If needed, the cells were diluted to 700-1,000 cells/µl in condition media or 1X PBS containing 0.04% BSA. Once diluted, ~5000 cells were captured on the 10X Genomics Chromium platform using the 3′ transcriptome protocol (V3). After confirmation of efficient cDNA synthesis, samples were processed for Illumina sequencing and quality checked using the Agilent Fragment Analyzer and Qubit fluorescence (Invitrogen). Libraries were pooled to 10 nM and final concentrations were determined using the Kapa Biosystems Universal qPCR system. Pooled libraries were diluted and denatured to 250 pM and run on the NovaSeq 6000 SP flow cell (28 × 91).
5
Cultivation of Feline CD4+ T Cells
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Mya-1 feline CD4+ T cells were cultured in RPMI 1640 medium with 2 mM l -glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, 10% FBS, and 1% penicillin and streptomycin and supplemented with 0.05 mM 2-mercaptoethanol and 100 units/mL recombinant human IL-2 (R&D Systems, Minneapolis, MN, USA). Cultures were maintained by the addition of fresh medium to cells every 2–3 days, maintained at 37 °C in a humidified atmosphere containing 7% CO2. Cells were stained with Trypan Blue and counted with Luna II cell counter (Logos Biosystems) for cell viability assessment. The viability was always found to be ~80%.
6
Metformin-Resistant Cancer Cell Assay
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A549, H460, HeLa, and MDA-MB-231 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in growth medium (RPMI1640 with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic) (Thermo Fisher Scientific). A549-W cells were cultured with metformin to establish A549-R. The concentration of metformin was increased by 0.5 mM every 2 weeks until it reached 8 mM, which is half of the maximal inhibitory concentration (IC50) for metformin in A549-W cells. The cells were seeded in 96-well plates and incubated under the indicated experimental conditions for the cell viability assay. The MTT reagent (CellTiter 96®, Promega) was added to each plate and incubated for 4 h at 37 °C according to the manufacturer’s instructions. Absorbance at 470 nm was detected using a MULTISKAN SkyHigh spectrophotometer (Thermo Scientific). The cells were seeded in 6-well plates and counted by trypan blue (Thermo Fisher Scientific) exclusion assay using a LUNA-II™ cell counter (Logos Biosystems) to assess the cell growth rates.
7
Isolation of Immune Cells from Salivary Gland
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For the isolation of immune cells from the salivary gland, a lateral salivary gland lobe was minced into 1–3 mm pieces and was digested with collagenase (1 mg/mL, FUJIFILM Wako Pure Chemical Corp.), hyaluronidase (1 mg/mL, Sigma-Aldrich Co. St. Louis, MO, USA), and DNase (10 μg/mL, Roche Diagnostics K.K., Tokyo, Japan) in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C for 40 min using the gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Next, splenocytes were homogenized in DMEM containing 2% FBS using a gentleMACS Dissociator (Miltenyi Biotec). Red blood cells were removed from the spleen cells using 0.83% ammonium chloride, and the viability and number of the isolated cells were evaluated on a Luna II cell counter (Logos Biosystems, Gyeonggi-do, Korea) using trypan blue staining. Subsequently, a proportion of the suspended cells was analyzed by flow cytometry. The absolute number of immune cells was calculated using data on total cell number and proportion of each cell type. As for the salivary gland, we used lateral lobe to determine the cell number and the proportion of immune cells.
8
Quantification of Neutrophil Extracellular Traps
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NETs were induced from bone-marrow derived neutrophils and quantified according to a previous study [24 (link)] with slight modifications. Briefly, bone marrow cells were harvested from the femur and tibia of mice, and then purified using a three-phase Percoll gradient (55%, 60%, 80%). Neutrophils at the 60%/80% Percoll interface were isolated, washed, and counted using the LUNA-II cell counter (Logos Biosystems, South Korea). Immediately after seeding neutrophils in a black 96-well plate (1 × 105 cells per well), NETosis was induced by adding 30 µL of PMA (2 µM stock) per well. After 3 h of incubation, free DNase-I (Invitrogen, 5 U) or DNase-NZ (5 U) was added to degrade NETs. After a further 45 min, 30 µL of SYTOX Green (50 µM stock) was added to each well and incubated for 15 min to stain extracellular DNA. Fluorescence (Ex. 488 nm / Em. 523 nm) was measured and compared using the SpectraMax iD3 microplate reader (Molecular Devices, USA). The percentage of DNA released by PMA was calculated using the following formula:
9
BALF Cell Enumeration Protocol
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The cell number and albumin concentration in BALF was determined immediately after collection. The BALF was centrifuged at 1500 rpm/min for 10 min,and the cell pellet used for determination of cell number while the supernatant was retained for ELISA assays.The cell pellet was resuspended in 200ul PBS solution, and then detected with LUNA-II cell counter (Logos Biosystems, Korea).
10
Single-cell analysis of zebrafish kidney
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Three male fish per genotype were used for our studies for robust biological replication, resulting in a total of 6 samples. Since we were limited by the number of fish we could transport, male fish were used to avoid confounding effects of presence/absence of egg clutches between female fish on the day of dissection. Head regions of the kidney, which contains the kidney marrow and constitutes the primary site of hematopoiesis in adult zebrafish were dissected from each fish. Care was taken to avoid inclusion of blood vessels as lysis of red blood cells can lead to contamination of ambient RNA with hemoglobin (Hb) genes in droplet-based single cell sequencing methods. Dissected kidneys were transferred into 2 ml Eppendorf tubes containing 500 µL of modified 1X phosphate buffer saline (PBS) supplemented with 0.5% bovine serum albumin and 1% Penicillin-Streptomycin. Single cell suspensions were obtained by dissociating tissues with 200 µL pipette tips and a 20-gauge needle, followed by washing with PBS and filtering through a 40 µm cell strainer (Corning). Cells were counted and viability assessed using a Luna II cell counter (Logos Biosystems) with trypan blue, and dead cells were removed using a Dead Cell Removal kit (StemCell).
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