CHO cells cultured semi-confluently beforehand in a 100 mm culture dish were trypsinized. In 24-well plates, 40 000 CHO cells were seeded per well and cultured for one day. After the culturing, the medium was removed by an aspirator and the cells were washed twice with phosphate buffer saline (PBS)(−), and this was followed by the addition of a drug (250, 625, 1250, 2500, 5000 μM) dissolved in PBS(−). The plate was left in the incubator for 30 min. After that, PBS (−) solutions were removed with an aspirator and the cells were washed twice with fresh PBS(−); the cells were then released by trypsin treatment, suspended in αMEM (10% FBS), and the cell number was measured using a Coulter Counter Z1. The cell suspension was put into a 24-well plate (5000 cells/well). After culturing for 3 days, the medium was removed by an aspirator and the cells were washed twice with PBS(−). The washed cells were released by trypsin treatment and suspended again in PBS(−), followed by measurement of the cell count using a Coulter Counter Z1. The cell growth rate was calculated by standardizing the obtained cell count against the cell count obtained without addition of the drug [25 ].
Z1 coulter counter
Manufactured by Beckman Coulter
Sourced in United States, Japan, Canada
The Z1 Coulter Counter is a laboratory instrument used for the automatic counting and sizing of particles suspended in an electrolyte solution. The device measures the changes in electrical resistance as particles pass through a small aperture, providing accurate particle counts and size distributions.
Automatically generated - may contain errors
Lab products found in correlation
Inverted phase contrast microscope, by Olympus (3 mentions)
Quick imaging system, by Olympus (3 mentions)
Gibco trypan blue solution 0.4, by Thermo Fisher Scientific (2 mentions)
Lympholyte m cell separation media, by Cedarlane (2 mentions)
Gefitinib, by Selleck Chemicals (2 mentions)
Ym155, by Selleck Chemicals (2 mentions)
Bi2536, by Selleck Chemicals (2 mentions)
Accutase solution, by GE Healthcare (2 mentions)
Sbiii, by Merck Group (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Nonhuman primate cd11b microbeads, by Miltenyi Biotec (2 mentions)
Bovine serum albumin (bsa), by Miltenyi Biotec (2 mentions)
Trypan blue solution, by Merck Group (2 mentions)
Xcelligence system, by Agilent Technologies (1 mentions)
Nec854010uc, by PerkinElmer (1 mentions)
Ls6500 scintillation counter, by Beckman Coulter (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
99 protocols using z1 coulter counter
1
Cytotoxicity Assay of Drug Compounds
2
Isolation of Tumor-Infiltrating Cells
3
Antimony Resistance in Leishmania infantum
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This study used lines of L. infantum (MHOM/BR/74/PP75) wild type (LiWTS) and resistant to potassium antimonyl tartrate (SbIII) (LiSbR). The resistant line (LiSbR) was previously selected in vitro by a step-wise increase of SbIII drug pressure [29 (link)]. These parasites were further maintained in culture under SbIII pressure, and the effective concentration required to decrease growth by 50% (EC50) was determined using a model Z1 Coulter Counter (Beckman Coulter, Fullerton, CA, USA). EC50 values for LiWTS and LiSbR obtained in this study were 0.12 mg/ml and 1 mg/ml, respectively, with an eight-fold resistance index (data not shown). Then, promastigote forms of these lines were grown at 26 °C in M199 medium supplemented with 40 mM HEPES pH 7.4, 1 μg/ml biotin, 5 μg/ml hemin, 2 μg/ml biopterin, 2 mM l -glutamine, 500 U penicillin, 50 μg/ml streptomycin and 10% (v/v) heat-inactivated fetal bovine serum [29 (link)]. Three independent biological replicates of each line were cultured. Based on previous studies of our group [29 (link)], wild-type L. infantum parasites were incubated for 24 h in the absence of drug (LiWTS 0), and resistant parasites were treated with 0.06 mg/ml SbIII (LiSbR 0.06), which corresponds to half of the SbIII IC50 for the LiWTS line. Cells were washed in RPMI medium, pelleted by centrifugation and frozen at − 70 °C.
4
Real-time Cell Proliferation Assay
5
Lung Inflammation Induction in Mice
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Male Institute of Cancer Research (ICR) mice (6–8 weeks old) were purchased from the National Laboratory Animal Centre (Taipei, Taiwan) and handled according to the guidelines of Animal Care Committee of Chang Gung University (Approval Document No. CGU 16-046) and National Institutes of Health (NIH) Guides for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). ICR mice were anesthetized and individually placed on a board in a near-vertical position and the tongues were withdrawn with lined forceps. TNF-α (0.25 mg/kg body weight) was placed posterior in the throat and aspirated into the lungs. Control mice were administrated with sterile 0.1% bovine serum albumin (BSA). Mice regained consciousness after 15 min. Mice were administered a dose of MVS (0.1 mg/kg body weight) for 24 h before TNF-α treatment, and sacrificed after 24 h. Bronchoalveolar lavage (BAL) fluid was collected through a tracheal cannula using 1 mL aliquots of ice-cold PBS solution. Leukocyte count was determined by a Z1 Coulter Counter (Beckman Coulter, Indianapolis, IN, USA) as previously described [33 (link)].
6
Methionine and Cystine Uptake Assays
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For methionine uptake assays, cells were cultured and labeled as described above for the protein synthesis assay, were washed three times in cold PBS, and lysed in Triton lysis buffer. For cystine uptake assays, cells were treated the same but labeled for the final 10 min with medium containing 0.1 µCi L-[1, 2, 1', 2'-14C]-Cystine (PerkinElmer, NEC854010UC) and washed three times in ice-cold PBS containing cold cystine (1 mM), prior to lysis in Triton lysis buffer. Whole-cell radiolabel incorporation was quantified with a Beckman LS6500 scintillation counter. Cells from identically treated parallel plates were counted using a Beckman Z1-Coulter Counter to normalize uptake measurements to cell number.
7
Milk Somatic Cell Isolation Protocol
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Milk samples were collected from a nearby dairy farm. 100 mL volumes of milk were hand-milked from a single quarter in each of 5 Holstein-Friesian cows identified as having a high somatic cell count at the most recent routine count. All samples were collected midway through milking. Samples were processed following the methodology used by Piepers et al.[51 (link)]. 25 mL volumes of milk were diluted 50:50 in cold phosphate-buffered saline (PBS) (Life Technologies, Dublin, Ireland) before centrifuging at 300 × g for 15 minutes at 4°C. Supernatants and fat layers were discarded and cell pellets resuspended in 10 mL of cold PBS. Samples were centrifuged at 4°C for 10 minutes at 200 × g before washing the cells once more. Cells were counted using a Z1 Coulter Counter (Beckman Coulter, Co. Clare, Ireland).
8
Lipid Hydroperoxide Quantification in Cancer Cells
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A Cayman Lipid Hydroperoxide Assay Kit was used to determine total lipid hydroperoxides in cell samples. Cal27 cells were treated with MSA for 72 h. Following treatment, cells were collected by trypsinization and counted. Total lipid extracts were obtained and analyzed as recommended by the manufacturer in glass cuvettes on a Beckman DU650 spectrophotometer. Lipid hydroperoxides per cell was quantified by construction of an appropriate standard curve and normalized to cell number, as determined by a Z1 Coulter Counter (Beckman-Coulter, Brea, CA, USA).
9
DNA-based Cell Counting in Dynamic Culture
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DNA-based cell counts were performed according to previous studies [11] (link), [12] (link), [13] for dynamic cultivation.
Total DNA was quantified with the NanoDrop 1000 Spectrophotometer (ND-1000, Thermo Fisher Scientific, Massachusetts, USA). Finally, cell numbers were calculated using a previously constructed working curve based on cell numbers determined with the Z1 Coulter Counter (Beckman Coulter, California, USA) and total DNA.
The mean DNA-based cell count of the three areas under dynamic cultivation was compared with that of a single collagen sheet under static cultivation.
Total DNA was quantified with the NanoDrop 1000 Spectrophotometer (ND-1000, Thermo Fisher Scientific, Massachusetts, USA). Finally, cell numbers were calculated using a previously constructed working curve based on cell numbers determined with the Z1 Coulter Counter (Beckman Coulter, California, USA) and total DNA.
The mean DNA-based cell count of the three areas under dynamic cultivation was compared with that of a single collagen sheet under static cultivation.
10
Cell Proliferation Monitoring Protocols
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