MCF7 cells were plated in normal medium (DMEM, 10% FCS, L-glutamine, supplemented with Pen-Strep) for 24-hr. Cells were then treated, initially for 7 days with doxycycline, with media changed every 3 days. For subsequent experiments, cells were treated for 72-hr. For radiation experiments, monolayer cells pre-treated with doxycycline, were exposed to increasing levels of radiation, and the cells collected by trypsinization and centrifugation. To quantitatively determine cell growth, the number of cells remaining after treatment was counted using an automatic cell counter (Biorad), compared to untreated cells and expressed as fold-change. To assess cell viability, cells were incubated for 1 minute with Trypan Blue (Sigma, #T8145) using a 1:1 ratio. The number of Trypan Blue positive cells (non-viable) was measured using an automatic cell counter (Biorad) and compared to untreated controls. Cells were also plated into mammosphere cultures to assess stem cell-like activity with no further drug treatment. All experiments were performed in triplicate and repeated at least three times independently.
Automatic cell counter
Manufactured by Bio-Rad
Sourced in United States
The Automatic Cell Counter is a laboratory instrument designed for the automated counting and analysis of cells. It provides rapid and reliable cell counts by using advanced optical detection technology. The core function of the Automatic Cell Counter is to accurately enumerate cells in a sample, without the need for manual counting methods.
Automatically generated - may contain errors
Lab products found in correlation
Trypan blue, by Merck Group (3 mentions)
Atcc 1174 liquid medium, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Amicon ultra, by Merck Group (1 mentions)
Trypan blue, by Bio-Rad (1 mentions)
Anti mouse antibodies, by BD (1 mentions)
Facscanto 2 cell analyzer, by BD (1 mentions)
Dnase, by Merck Group (1 mentions)
Mab160, by R&D Systems (1 mentions)
Mab392, by R&D Systems (1 mentions)
Mab266, by R&D Systems (1 mentions)
Mab672, by R&D Systems (1 mentions)
Transwell plate, by Corning (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Trypsinized, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
29 protocols using automatic cell counter
1
Doxycycline-Mediated Growth Inhibition in MCF7 Cells
2
Conditioned Medium from UB-tip Cells
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CM was prepared from a mean of 1 × 108 UBtip cells grown at 90–95% confluency in cell-culture dishes (Greiner, Cellstar (#639,160)) or pUBs grown in similar conditions. Cell cultures were washed twice with 1× PBS before a 24 h incubation with 25 mL EV-free medium [22 ] at 37°C in 5% CO2. EV-free medium is DMEM/F12 supplemented with 1 or 10% FBS. The medium was prepared according to Witwer et al. 2006 [26 ] as a 20% stock solution as follows: DMEM/F12 supplemented with 20% FBS is centrifuged for 24 h at 100 000 g and subsequently diluted out (to contain 1 or 10% FBS) and filtered through a 0.2 µm filter (Whatman). Residual EV contamination was not found, since no EV markers were found when applied to a Western blot as a control. Following the collection of the CM, cell cultures were trypsinized, the cells were counted, and cell viability was measured on an Automatic Cell Counter (BioRad) using a 0.1% trypan blue exclusion test.
The CM from pUB cells was harvested after 24–48 h of cell culture. Subsequently it was concentrated by filtration (Amicon Ultra, Millipore, 100K filters) from ~5 mL to 350 µL, and stored at −20°C until usage.
The CM from pUB cells was harvested after 24–48 h of cell culture. Subsequently it was concentrated by filtration (Amicon Ultra, Millipore, 100K filters) from ~5 mL to 350 µL, and stored at −20°C until usage.
3
Growth of Dunaliella tertiolecta in Batch Culture
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Strain LB-999 of D. tertiolecta was obtained from the UTEX Culture Collection of Algae (University of Texas, Austin, TX, USA). Dunaliella tertiolecta cells were grown in a batch system in a sterile ATCC-1174 liquid medium (American Type Culture Collection, Manassas, VA, USA) containing 0.5 M NaCl on a rotary shaker at 25 °C, under a 14 h light/10 h dark regime (50 μmol photons m−2 s−1). Cell densities were measured using an automatic cell counter (Bio-Rad Laboratories, USA). For the purpose of clarity, all experiments were performed in independent biological/technical triplicates and began at equal cell densities for standardization unless otherwise stated.
4
Enzymatic Digestion and Flow Cytometry Analysis
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After 15 days of the biocurative application, the samples were immediately cut using surgical scissors and submitted to an enzymatic digestion solution containing collagenase IV (40 U/mL) and DNAse (Sigma, 40 μg/mL) for 30 min at 37 °C, 200 rpm. Then, the samples were filtered through cell strainers of 70 μm and 40 μm. The resulting cell suspension was counted in an automatic cell counter (BioRad) and 0.5 × 106 cells from each mouse were incubated with anti-mouse antibodies (BD Biosciences) conjugated to fluorochromes for surface molecules. The conjugated antibodies used were CD45 (PE-Cy7), FcεRI (FITC), CD117 (APC), F4/80 (PerCP), CD11c (FITC) and CD206 (APC). The labeled cells were acquired on the FACSCanto II Cell Analyzer (BD Biosciences). The dispersions were analyzed using the FlowJo software (BD Biosciences).
5
Chemotaxis Assay for CXCR3-Mediated Migration
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Chemotaxis assay was performed using 24-well Transwell plates (Costar, 3415, USA). One milliliter supernatants from ME180 cells which were mock-infected or infected with HSV-2, or mock-transfected or transfected with ICP4 expressing plasmid were added to the lower chamber. To examine the roles of CXCR3 and CXCR3 ligands in mediating cell migration, supernatants or cells were incubated with anti-CXCL9 (10 μg/mL, R&D Systems, MAB392, USA), -CXCL10 (2 μg/mL, R&D Systems, MAB266, USA), -CXCL11 (2 μg/mL, R&D Systems, MAB672, USA) or -CXCR3 (1 μg/mL, R&D Systems, MAB160, USA) neutralizing Abs, respectively, for 1 h, according to the manufacturer's instructions. Activated PBMCs and CD4+ T cells (5 × 105) in 100 μL RPMI-1640 medium were added to the upper chamber. The chambers were incubated for 2 h at 37°C in a 5% CO2 incubator. Cell migrated to the lower chambers were collected and counted using an automatic cell counter (Bio-Rad).
6
HL60 Cell Culture and Vincristine Resistance
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HL60 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Invitrogen) supplemented with 20% fetal bovine serum (FBS, Sigma), 1% l -glutamine, penicillin (100 U/mL, Invitrogen), and streptomycin (100 mg/mL, Invitrogen). Cell number was monitored daily and culture was maintained at a density of 2 × 105–2 × 106 cells/mL. Cell number was monitored daily using a BioRad automatic cell counter and viability was assessed by trypan blue exclusion (0.4% supplied with the cell counter slides and added to the cells in a 1:1 v/v ratio according to manufacture’s instructions). HL60-resistant cells were supplemented with the respective concentration (10 or 100 nM) of vincristine (in H2O, Sigma). The cells were passaged every 2–3 days and drug was added freshly each time.
7
Inducible p130 Cell Proliferation
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NHF-1 inducible p130 cells were seeded in 10 cm plates, and p130 WT or AA was induced by the addition of 100 ng/ml doxycycline (or DMSO as a control) to media. Cells were trypsinized (Gibco), counted with an automatic cell counter (Bio-Rad), and replated in fresh doxycycline-containing media every 48 hr for 2 weeks. Cell count was plotted versus time, differences were assessed using a Student’s t-test, and cell count was used to calculate doubling times. Experiments were carried out in triplicate and repeated three times.
8
Cell Proliferation Measurement Techniques
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Cell proliferation was assessed as described (Tsitsipatis et al., 2021 (link)). Briefly, 2 × 104 cells were plated per well and proliferation rates were assessed using both cell counting and BrdU incorporation up to day 6. For cell counting, cells were harvested and washed once with 1× PBS (Gibco), and total cells were counted on an automatic cell counter (Bio‐Rad). For BrdU incorporation, BrdU Cell Proliferation Assay (Cell Signaling Technology) was employed following the manufacturer's instructions. Briefly, BrdU was added one day after seeding and the incorporation was detected for up to day 6 by reading at 450 nm on a GloMax Explorer plate reader (Promega).
9
Cytoprotective Drug Screening Protocol
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Cells were seeded in 12 well plates that were irradiated with UV in PBS and after UV, the PBS was immediately replaced with media containing the test drugs. For H2O2 treatment, the cells were pretreated with drugs for 2 h before adding H2O2. The plate was further incubated with the drugs for 24 h before the cells were collected and mixed with a 1:5 dilution of Trypan blue (Gibco, Gaithersburg, MD, USA). The number of viable cells was determined by counting the non-stained cells using dual chamber cell counting slides and an automatic cell counter (BioRad, Hercules, CA, USA).
10
Multiparameter Flow Cytometry Analysis
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The cells in the exudate were counted on an automatic cell counter (Bio-Rad) using trypan blue to assess viability. The cells (1 × 106) were suspended in FACS buffer (PBS, 2% (v/v) fetal calf serum, and 0.05% (w/v) NaN3), blocked with Fc block (anti-CD16/32) and then stained with lineage specific and fluorochrome labelled monoclonal antibodies against T cells (CD3-FITC), B cells (B220-PE), Monocytes (CD11b-APC), Dendritic cells (CD11c-PerCP), Macrophages (F4/80-PE-Cy7) and Neutrophils (Ly6G-APC-Cy7). The cells were washed with FACS buffer and then 50,000 cells were acquired in Flow cytometer (FACS caliber, BD, USA). The data was analyzed by FLOW JO software (Tree star Inc.).
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