Automated cell counter

Manufactured by Countstar

Sourced in China, United States

The Automated Cell Counter is a compact, high-precision laboratory instrument designed to accurately count and analyze cells. The device utilizes advanced optical and digital technologies to provide reliable cell count and viability measurements, making it a versatile tool for various applications in the life sciences and biomedical research.

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Cell counter (28 citations) Countess II FL Automated Cell Counter (16 citations) Countess® II Automated Cell Counter (10 citations) Countess™ automated cell counter (7 citations) FL Automated Cell Counter (6 citations) Automated cell counter system (2 citations) Automated Cell Counter IM1200 (2 citations) Automated Cell Counter machine (2 citations) II Automated Cell Counter (2 citations) Automated Cell Counter (IC1000, (2 citations)

102 protocols using automated cell counter

Quantification of Nuclei Extracted from Human WAT

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Timing: 15 min

The following steps detail how to stain and count the number of nuclei extracted.

24.

Add 1 drop of ReadyProbes NucBlue (Hoechst 33342) to the 500 μL nuclei solution and leave on ice protected from light for 15 min.

Note: It is possible to count the nuclei extracted using DAPI under a microscope.

25.

Mix nuclei solution with pipetting before immediately adding 10μL of nuclei solution to countess slide.

26.

Count nuclei with Countess 3 Automated Cell Counter (Figure 3).Figure 3

Isolated Nuclei from human WAT

Nuclei isolated from human subcutaneous WAT and stained with NucBlue and counted with Countess 3 Automated Cell Counter. Image is jpg readout from the Countess 3.

Note: Typical yield is 180K nuclei at a concentration of 360K/mL.

27.

To ensure accurate nuclei loading on the ICELL8 CX, we recommend diluting the sample with DPBS (−/−) to 200K/mL and re-counting for validation.

Whytock K.L., Divoux A., Sun Y., Hopf M., Yeo R.X., Pino M.F., Yu G., Smith S.R., Walsh M.J, & Sparks L.M. (2023). Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling. STAR Protocols, 4(1), 102054.

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Evaluating Cell Growth and Viability

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PCa cells were plated in 6-well plates in triplicate and allowed to adhere for 24Hrs prior to treatment with doxycycline (1μg/ml) or ATRA (Sigma-Aldrich, St. Louis, MO, USA). At the indicated time points, cells were trypsinized, pelleted, and resuspended in a known volume. Cell counts were established using a Countess automated cell counter. Live versus dead cells were quantified by trypan blue exclusion using a Countess automated cell counter. Cell growth was determined by normalizing cell numbers at the indicated time points to the number of cells plated. Doubling times were determined using relative growth values from T24 and T96 time points.
The viability of cells expressing CD38 treated with FK866 was determined using Cell Titer-Glo 2.0 (Promega, Madison, WI, USA). Briefly, cells treated with vehicle or doxycycline (1μg/mL) were plated in 96-well plates (N=7) and allowed to adhere for 8Hrs prior to the addition of FK866. Luminescence was detected using a FLUOstar Optima spectrometer (BMG Labtech, Cary, NC, USA) and viability was determined relative to control.

Chmielewski J.P., Bowlby S.C., Wheeler F.B., Shi L., Sui G., Davis A.L., Howard T.D., D’Agostino RB J.r., Miller L.D., Sirintrapun S.J., Cramer S.D, & Kridel S.J. (2018). CD38 Inhibits Prostate Cancer Metabolism and Proliferation by Reducing Cellular NAD+ Pools. Molecular cancer research : MCR, 16(11), 1687-1700.

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Mineral Mixture Cytotoxicity on 3T3 Fibroblasts

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The viability of 3T3 mouse fibroblasts exposed to the antibacterial mineral mixtures was measured to determine if mammalian cells could withstand the application of a mineral poultice while maintaining antibacterial characteristics. The antibacterial mineral mixtures were prepared as described previously with 95% F-hectorite clay, 5% pyrite and exchanged with 30 mM FeSO₄ and autoclaved prior to testing. All experiments were performed in a 12-well plate using 3T3 mouse fibroblast cells grown to 85% confluency in RPMI media supplemented with 10% dialyzed fetal bovine serum. The fibroblast cells were reacted with 25 and 100 mg/mL concentrations of the antibacterial mineral mixtures in RPMI media. After 24 h of exposure, the media and minerals were decanted and the cells were rinsed with 0.8% sterile sodium chloride buffer three times to remove excess minerals. The cells were then trypsinized to detach them from the 12-well cell culture treated plate. Samples were then pelleted in a 1.5 mL centrifuge tube and stained with trypan blue to determine cell viability using a Countess automated cell counter. A control well with no cells and 100 mg/mL minerals was also included to determine if the minerals interfered with the viability assay.

Morrison K.D., Martin K.A., Wimpenny J.B, & Loots G.G. (2022). Synthetic antibacterial minerals: harnessing a natural geochemical reaction to combat antibiotic resistance. Scientific Reports, 12, 1218.

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Cell Growth Kinetics under Normoxia and Hypoxia

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100,000 of each indicated control and knockdown cells were seeded in 24 well plates on day 0, triplicate wells of each group were trypsinized and the cell numbers were counted using Countess II Automated Cell Counter on each indicated timepoint. The regular cultured experiments were performed for day 0, 2, 4 and 6. The hypoxic experiment was performed in 3% O₂ for day 0, 2 and 4.

Deng Q., Natesan R., Cidre-Aranaz F., Arif S., Liu Y., Rasool R.U., Wang P., Mitchell-Velasquez E., Das C.K., Vinca E., Cramer Z., Grohar P.J., Chou M., Kumar-Sinha C., Weber K., Eisinger-Mathason T.S., Grillet N., Grünewald T, & Asangani I.A. (2022). Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1. Cell reports, 39(11), 110971.

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In Vitro Cytotoxicity Assay for Potent Compounds

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In vitro toxicity studies of the potent
compounds were accomplished according to our reported modified procedure.^{17 (link),24 (link)} HEK293 cells were counted using a Countess automated cell counter,
and 4000 cells per well were seeded in black 96-well plates. Compounds
were serially diluted starting at 50 μg/mL, added in triplicate,
and incubated for 24 h. Resazurin solution (5%) for a final volume
of 240 μL in each well was added after 24 h. After the addition
of 5% resazurin, plates were incubated for 4 and 6 h before taking
the readings for cell viability. Fluorescence was measured at 544
nm (excitation) and 590 nm (emission) using a BMG Labtech Fluostar
Optima plate reader.

Whitt J., Duke C., Ali M.A., Chambers S.A., Khan M.M., Gilmore D, & Alam M.A. (2019). Synthesis and Antimicrobial Studies of 4-[3-(3-Fluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic Acid and 4-[3-(4-Fluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic Acid as Potent Growth Inhibitors of Drug-Resistant Bacteria. ACS Omega, 4(10), 14284-14293.

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Repeated CAR T-Cell Coculture Expansion

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Either 5 × 10⁵ or 1 × 10⁶ sorted CAR T cells were cocultured in cytokine-free Opt5 media at a 1:1 ratio with gamma-irradiated K562-CD19 cells or gamma-irradiated K562-mesothelin cells, with a total cell concentration of 1 × 10⁶ per ml. After 7 days, cells were counted with a Countess automated cell counter, gating for cells between 3 and 15 μm in diameter. Then, 5 × 10⁵ cells were reseeded in 1 ml of fresh Opt5 with 5 × 10⁵ target cells. This process was repeated on days 12, 17, 22, and 27. At each time point, cell count, size, and viability were recorded.

Collins S.M., Alexander K.A., Lundh S., Dimitri A.J., Zhang Z., Good C.R., Fraietta J.A, & Berger S.L. (2023). TOX2 coordinates with TET2 to positively regulate central memory differentiation in human CAR T cells. Science Advances, 9(29), eadh2605.

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Cell Seeding Density and Growth Kinetics

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Cells were seeded in six-well plates at 5.2 × 10³ cells/cm². Cells were trypsinized and counted over 4 days using a Countess Automated Cell Counter; 0 h is the time at plating (n = 3).

Tye C.E., Ghule P.N., Gordon J.A., Kabala F.S., Page N.A., Falcone M.M., Tracy K.M., van Wijnen A.J., Stein J.L., Lian J.B, & Stein G.S. (2022). LncMIR181A1HG is a novel chromatin-bound epigenetic suppressor of early stage osteogenic lineage commitment. Scientific Reports, 12, 7770.

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Generating Bone Marrow Chimeric Mice

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Bone marrow chimeric mice were generated as previously described by our laboratory (11 (link)). In brief, bone marrow was aseptically harvested from the tibias and femurs of eight-week-old B6.CD45.1 donor mice (n=18). Erythrocytes were lysed and the cells were counted using a Countess automated cell counter. Eight-week-old B6.CD45.2 mice (n=9) received a single 1000-cGy γ-irradiation dose using a Cs-137-based Gammacell 40 irradiator. The mice heads were shielded with a lead bar so as to deliver the irradiation to the body only. Six hours after shielded irradiation, busulfan (30 mg/kg) was administered to completely ablate the bone marrow of the recipient mice. Donor bone marrow (CD45.1) was transplanted twelve hours after busulfan ablation. Shielded bone marrow chimeras were maintained on antibiotics trimethoprim/sulfamethoxazole (40 mg/5 mg, respectively). Eight weeks after irradiation, 95% of the circulating monocytes were of donor origin (Fig. 1).

Makinde H.M., Just T.B., Gadhvi G.T., Winter D.R, & Schwulst S.J. (2019). Microglia adopt longitudinal transcriptional changes after traumatic brain injury. The Journal of surgical research, 246, 113-122.

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Isolation of Human Gastric Tissue Cells

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Human gastric tissue was obtained from patients and placed into a sterile RNase-free culture dish containing an appropriate amount of calcium-free and magnesium-free 1× phosphate-buffered saline (PBS) on ice. The tissue was then transferred into the culture dish, cut into 0.5 mm² pieces, and washed with 1× PBS, and blood residue and fatty layers were removed. Tissues were dissociated into single cells in dissociation solution. The overall cell viability was more than 85%, as confirmed by trypan blue exclusion. The single-cell suspensions were counted using the Countess II Automated Cell Counter, and the concentration was adjusted to 700–1200 cells/μl before single-cell analysis.

Zhao W., Jia Y., Sun G., Yang H., Liu L., Qu X., Ding J., Yu H., Xu B., Zhao S., Xing L, & Chai J. (2023). Single-cell analysis of gastric signet ring cell carcinoma reveals cytological and immune microenvironment features. Nature Communications, 14, 2985.

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Assessing Koumine's Impact on Tetrahymena

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T. thermophila cells were cultured in SPP in test tubes containing 5 mL of medium and koumine was added at 0, 0.05, 0.1, 0.2, 0.4 and 0.8 mg/mL. 0 mg/mL koumine was used as blank control and 0.1% DMSO was used as negative control (NC). Penicillin G (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (0.02 μg/mL) were then added to each tube of medium to prevent bacterial and fungal contamination. Cells were inoculated to a final density of 6250 cells/mL and tubes were incubated for 72 h at 30°C with constant shaking at 135 rpm [31 (link)]. Cells were counted using an automated cell counter (Countstar, Shanghai, China). Cell viability was determined at 24 h using a Cell Counting Kit-8 (CCK-8) (Nanjing Jiancheng, Nanjing, China) according to the manufacturer’s instructions [32 (link)]. The oxidative stress, apoptosis, DNA damage, ultrastructural changes and gene expression effects was determined after T. thermophila exposed to various levels koumine at 24 h. The specific experimental process was shown below.

Ye Q., Zhang C., Wang Z., Feng Y., Zhou A., Xie S., Xiang Q., Song E, & Zou J. (2019). Induction of oxidative stress, apoptosis and DNA damage by koumine in Tetrahymena thermophila. PLoS ONE, 14(2), e0212231.

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