The following cell lines were used: CT-26 murine colon carcinoma (ATCC), 4T1 murine mammary carcinoma (ATCC), MDA-MB-231 human breast adenocarcinoma (ECACC), COLO201 human colorectal adenocarcinoma (ATCC), HepG2 human hepatocellular carcinoma (State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Russia); A549 human non-small-cell lung carcinoma, SK-BR-3 human breast adenocarcinoma, SNU-1 human gastric carcinoma, Jurkat human acute T-lymphoblastic leukemia, and WI-38 human lung fibroblasts (normal cells) obtained from the Bioresource Collection of Cell Lines and Primary Tumors of the Blokhin National Medical Research Center of Oncology (Moscow, Russia). Cells were counted on a Countess® II FL automatic cell counter (Thermo Fisher Scientific, Singapore).
Countess 2 fl automatic cell counter
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Countess II FL Automatic Cell Counter is a compact, automated device designed for accurate cell counting and viability analysis. It utilizes automated fluorescence microscopy to provide precise cell counts and viability measurements for a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Erythrocyte depletion kit, by Miltenyi Biotec (1 mentions)
Macsxpress eosinophil isolation kit, by Miltenyi Biotec (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Easysep human cd4 cd127lowcd25 regulatory t cell isolation kit, by STEMCELL (1 mentions)
Countess 2 fl, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by BD (1 mentions)
Apc conjugated anti cd44 antibody, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
9 protocols using countess 2 fl automatic cell counter
1
Comprehensive Cell Line Panel for Cancer Research
2
Isolation of Eosinophils from Monkey Blood
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EOS were purified from 30 mL monkey peripheral blood samples though an immunomagnetic negative-selection approach with the MACSxpress Eosinophil Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and the residual erythrocytes were removed with the Erythrocyte Depletion Kit (Miltenyi Biotec). Then, the number of harvested EOSs suspended in PBS was quantified on a Countess II FL automatic cell counter (Thermo Fisher Scientific).
3
Oyster Gonad Nuclei Extraction
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Oyster gonad tissues were minced to 1 mm3 and stored at -80°C prior to nuclei extraction. Following histological confirmation, the nuclei from male and female gonadal cells at stage Ⅰ were isolated and resuspended in 2 mL of phosphate-buffered saline containing 0.01% bovine serum albumin. The concentration of nuclei was determined using Thermofisher Countess II FL Automatic Cell Counter and was adjusted as needed to achieve the ideal range for loading on the 10X Chromium chip.
4
Cytotoxicity of Dox and Au-GSH-Dox
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First, 5 × 105 cells (D17 and U2OS) per well were seeded in a 6-well plate. After 24 h of standard incubation, the media were removed, and the tested compounds were introduced to the cells at increasing concentrations: 5, 10, 20, and 50 μg/mL for Dox and Dox in Au-GSH-Dox and 100, 200, 400, and 1000 μg/mL for Au-GSH. No substances were added to the medium of the control group. The cells were incubated with the tested substances for 24 h, then harvested by trypsinization and centrifuged at 1800 rpm for 3 min. After this, 10 μL of the cell suspension was mixed with 10 μL of 0.4% Trypan Blue and cell counts were evaluated with a Countess II FL Automatic Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA). Dead cells were stained blue, and cell mortality was expressed as the percentage of dead cells (from the total cell number).
5
Cell Viability Assay After Irradiation
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At 1–4 days after irradiation, the medium was removed, and the cells were washed with PBS (2.0 mL) three times. Cells were detached by incubating in trypsin–EDTA (0.20 mL) (Thermo Fisher Scientific) for 5 min, and then, the reaction was stopped by applying medium (0.20 mL). The cell suspension was pipetted thoroughly, and the viable cells were counted by 0.4% trypan blue (Thermo Fisher Scientific) staining using the Countess-II FL automatic cell counter (Thermo Fisher Scientific).
6
Isolation of Human PBMC Subsets
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To prepare peripheral blood mononuclear cells (PBMCs), diluted sodium heparinized whole blood, approx. 30-40 mL was layered on Ficoll-Paque Density Gradient Medium (ThermoFisher Scienti c) and centrifuged for 40 min at 400 x g with low acceleration and no brake. PBMCs were collected from the interphase and thereafter washed with PBS. The cells were then resuspended in PBS containing 2 mM ethylenediaminetetraacetic acid (EDTA), and 0,5% fetal bovine serum (FBS). Cells were counted using a Countess II FL Automatic Cell Counter (ThermoFisher).
To isolate the T reg and T conv cell fractions, an EasySep Human CD4 + CD127 low CD25 + Regulatory T Cell Isolation Kit (STEMCELL Technologies) was used according to manufacturer´s instructions on PBMCs resulting in two cell fractions; T reg cells de ned as CD4 + CD127 low CD25 + and T conv cells de ned as CD4 + CD127 + CD25 -T cells.
Following isolation, all cells were resuspended in complete RPMI (Fisher Scienti c) cell medium with 10% FBS (Fisher Scienti c) and 2% Penicillin Streptomycin solution (Fisher Scienti c).
To isolate the T reg and T conv cell fractions, an EasySep Human CD4 + CD127 low CD25 + Regulatory T Cell Isolation Kit (STEMCELL Technologies) was used according to manufacturer´s instructions on PBMCs resulting in two cell fractions; T reg cells de ned as CD4 + CD127 low CD25 + and T conv cells de ned as CD4 + CD127 + CD25 -T cells.
Following isolation, all cells were resuspended in complete RPMI (Fisher Scienti c) cell medium with 10% FBS (Fisher Scienti c) and 2% Penicillin Streptomycin solution (Fisher Scienti c).
7
Amoebicidal Activity Evaluation
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The amoebicidal activity experiments were carried out using an inverted light-microscope Leica DM IL (Wetzlar, Germany). Initially, the trophozoites were counted using a Countess II FL automatic cell counter (Thermo Fisher Scientific, Madrid, Spain) to prepare a working concentration of 5.10 4 trophozoites/well and 10 5 cells/well, respectively, for Acanthamoeba spp. and Naegleria fowleri strains), and 50 µL per well was seeded in triplicate in a 96-well plate (Thermo Fisher Scientific, Madrid, Spain).
After that, a serial dilution of CLORICAN in the same culture medium was added to the plate (50 µL per well). As a negative control, the trophozoites were incubated with the medium alone. Plates were incubated with slight agitation for 96 h at 26 • C for Acanthamoeba spp. and 48 h at 37 • C for Naegleria. Subsequently, the plates were observed during the incubation time with an inverted light-microscope (15 min, 1 h, 24 h, 48 h and 96 h). The numbers of viable and nonviable trophozoite were counted using a Countess II FL automatic cell counter (Thermo Fisher Scientific, Madrid, Spain) at 96 h for Acanthamoeba spp. and 48 h for Naegleria fowleri using an inverted microscope and confirmed with trypan blue (0.4%) staining using the Countess II FL (Thermo Fisher Scientific, Madrid, Spain)
After that, a serial dilution of CLORICAN in the same culture medium was added to the plate (50 µL per well). As a negative control, the trophozoites were incubated with the medium alone. Plates were incubated with slight agitation for 96 h at 26 • C for Acanthamoeba spp. and 48 h at 37 • C for Naegleria. Subsequently, the plates were observed during the incubation time with an inverted light-microscope (15 min, 1 h, 24 h, 48 h and 96 h). The numbers of viable and nonviable trophozoite were counted using a Countess II FL automatic cell counter (Thermo Fisher Scientific, Madrid, Spain) at 96 h for Acanthamoeba spp. and 48 h for Naegleria fowleri using an inverted microscope and confirmed with trypan blue (0.4%) staining using the Countess II FL (Thermo Fisher Scientific, Madrid, Spain)
8
Identification of Mucoepidermoid Carcinoma CSCs
9
Enzalutamide and APOBEC3B Inhibitor Assay
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