ADSC at the third passage were collected by 0.25% trypsin-EDTA (Gibco, Grand Island, NY, USA), washed in PBS and resuspended in PBS at a concentration of 1 × 106 cells/ml. The cells were then blocked with 0.5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 20 min, followed by additional 30 min incubation at 4°C in the dark with one of the following antibodies: anti-CD44-FITC (1:50), anti-CD73-PE (1:50), anti-CD105-BV421 (1:100), anti-CD31-APC-Cy™7 (1:100), anti-CD45-APC (1:100), and anti-CD90-PerCP-Cy™5.5 (1:50). All antibodies were purchased from BD Biosciences (San Jose, CA, USA). The cells were subsequently washed in 1 ml 0.2% BSA and centrifuged at 500×g for 5 min. Antibody binding was detected using a Bio-Rad ZE5 cell analyzer (Bio-Rad, Hercules, CA, USA) and analyzed using the FlowJo® software.
Ze5 cell analyzer
Manufactured by Bio-Rad
Sourced in United States
The ZE5 Cell Analyzer is a flow cytometry instrument designed for high-throughput cell analysis. It features a compact design and is capable of analyzing a wide range of sample types, including cells, microbeads, and particles. The ZE5 provides users with precise and reliable data on various cellular parameters, such as size, granularity, and fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Human ab serum, by Gemini Bio (5 mentions)
Flow cytometry staining buffer, by Thermo Fisher Scientific (4 mentions)
Phytohemagglutinin (pha), by Roche (4 mentions)
Fcs express, by De Novo Software (3 mentions)
Liberase, by Roche (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Cd3 af700, by Thermo Fisher Scientific (3 mentions)
Cd4 bv605, by BD (3 mentions)
Cd137 apc, by BioLegend (3 mentions)
Ox40 pe cy7, by BioLegend (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (3 mentions)
Fcs express 6 flow cytometry, by De Novo Software (3 mentions)
Luna cell counter, by Logos Biosystems (2 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (2 mentions)
Fcs express flow cytometry software, by De Novo Software (2 mentions)
Fcs express 6 flow cytometry software, by De Novo Software (2 mentions)
Caspase 3 7 assay kit, by Thermo Fisher Scientific (2 mentions)
Fcs express multicycle, by De Novo Software (2 mentions)
Pe conjugated anti human hla a 03 clone gap a3, by BD (2 mentions)
Pe conjugated mouse igg2a isotype clone mopc 173, by BioLegend (2 mentions)
117 protocols using ze5 cell analyzer
1
Immunophenotyping of Adipose-Derived Stem Cells
2
Quantifying Oxidative Stress in RAW264.7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ROS production was estimated using an ROS Assay kit (Beyotime Institute of Biotechnology) as previously described (23 (link)). RAW264.7 cells (2x105 cells/ml) were cultured in a 24-well plates and incubated at 37˚C overnight. Cells were pretreated with different concentrations (0.1, 0.5 and 1.0 µg/ml) of TPs at 37˚C for 12 h, after which each well plate was exposed to 400 µM H2O2 at 37˚C for a further 12 h. Subsequently, the cells were washed three times with cold PBS and incubated with 5 µM DCFH2-DA (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C for 30 min. RAW264.7 cells were washed with DMEM culture medium and determined the fluorescent intensity was determined using a Bio-Rad ZE5 cell analyzer (Bio-Rad Laboratories, Inc.).
3
Hypoxia-Induced Apoptosis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AC16 cells were resuspended as 20,000 cells per ml in a tube and cultured under hypoxic conditions (1%O2/94%N2/5%CO2) or normal conditions for 48 h with three replicates for each experiment. Cells were stained with PI and Annexin V-FITC (Annexin V-FITC Apoptosis Detection Kit, Sigma-Aldrich) and sorted by flow cytometry using ZE5 Cell Analyzer (Bio-Rad).
4
Immunostaining of Adipose and Muscle Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six animals from each group were euthanized on day 60 for immunohistochemical analysis. To carry out immunohistochemical analyses, it was necessary to first deparaffinize and rehydrate the tendon tissue paraffin sections. Next, the standard protocol was followed by incubating the tissue cells, which were taken from both the abdominal muscle and adipose part of the rats, with an Immuno-Block reagent for 30 min. After that, the cells were treated with rat primary antibodies. These antibodies were polyclonal anti-C reactive protein antibody (anti-CRP; MBS4158517; 1:200; MyBioSource) and polyclonal anti-peroxisome proliferator-activated receptor gamma antibody (anti-PPAR-γ; MBS619780; 1:100; MyBioSource) [71 (link)]. After undergoing a counterstaining process with hematoxylin, the sections were properly dehydrated and fixed. The area of the positive signal was measured through the utilization of flow cytometry; specifically, the ZE5 Cell analyzer from Bio-Rad (Minneapolis, MN, USA). The calculation process was carried out with the aid of FlowJo® software version 10.9.
5
Assessing Cell Metabolism and Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) turnover was used to identify potential compound effects on cell metabolism and indirectly assay changes in cell number or viability. HEK Blue reporter cells were cultured and exposed to compounds as described above and 20 µl supernatant were collected for SEAP activity assays. U937 monocyte-like cells were cultured in RPMI-1640 containing 10% FBS, differentiated by addition of 100 nM PMA for 2 days and exposed to compounds at varying concentrations or solvent for 2 days. MTT dissolved in PBS was added to the cells to a final concentration of 1 mg/ml. Cells were then incubated at culture conditions for 1 h. Supernatants were removed after centrifugation at 400xg for 5 min and the formed formazan dye was dissolved in DMSO. Absorbance was measured at 570 nm and readings were normalized to solvent treated controls.
Exclusion of Helix NIR (BioLegend) was used to assay membrane integrity following compound treatment. Culture conditions for U937 were as described above, undifferentiated cells were incubated with varying concentrations of compound or with solvent. Helix NIR was added to the cells to a final concentration of 10 nM, cells were incubated at RT for 10 min and acquired on a ZE5 Cell Analyzer (Bio-Rad). The cutoff for positive staining was set to approximately the 99th percentile of unstained cells.
Exclusion of Helix NIR (BioLegend) was used to assay membrane integrity following compound treatment. Culture conditions for U937 were as described above, undifferentiated cells were incubated with varying concentrations of compound or with solvent. Helix NIR was added to the cells to a final concentration of 10 nM, cells were incubated at RT for 10 min and acquired on a ZE5 Cell Analyzer (Bio-Rad). The cutoff for positive staining was set to approximately the 99th percentile of unstained cells.
6
Quantitative Analysis of H2O2 and Polyamines in NP Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative H2O2 level in human NP cells (n = 5) was determined by a Hydrogen Peroxide Assay Kit according to the manufacturer's instructions. The AbGreen reacts with H2O2 in live NP cells and produces a green fluorescence. Fluorescence intensity was quantified using the Fiji package (https://imagej.net/software/fiji/ ). Besides, the H2O2 level in the tissue extracts of mouse disc (n = 5) was determined by another Hydrogen Peroxide Assay Kit (ab102500) according to the manufacturer's instructions. Total polyamine in the human NP tissues (n = 3) or cells (n = 5) was determined by the Total Polyamine Assay Kit. A fluorometric probe (Ex/Em = 535/587 nm) of H2O2 or polyamine was read out by a microplate reader (BMG LABTECH, Ortenberg, Germany).
The total ROS production in human and mouse NP cells (n =5) was determined using diacetyl dichlorofluorescein (DCFH-DA) staining and analyzed by flow cytometry (ZE5 Cell Analyzer, Bio-Rad, Hercules, USA) at a wavelength of 488 nm based on the manufacturer's instructions.
The total ROS production in human and mouse NP cells (n =5) was determined using diacetyl dichlorofluorescein (DCFH-DA) staining and analyzed by flow cytometry (ZE5 Cell Analyzer, Bio-Rad, Hercules, USA) at a wavelength of 488 nm based on the manufacturer's instructions.
7
Cell Cycle and Apoptosis Analysis Post-Radiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated and treated with a drug for one hour before radiation treatment. Following radiation (4 Gy), cells were harvested at predetermined time points. For cell cycle experiments, cells were fixed in 70% ethanol at 6-, 16-, and 24-h post-RT. Before analysis, fixed cells were resuspended in 1× PBS and stained with propidium iodide and RNase (Qiagen 19101). For analysis of apoptosis, cells were harvested by trypsinization 48 h after radiation and stained with annexin V and propidium iodide (Roche 11858777001) immediately preceding analysis. Both apoptosis and cell cycle samples were analyzed at the University of Michigan Flow Cytometry Core using the BioRad ZE5 Cell Analyzer (Supplementary Figs. 6 and 7 ). Data are shown as mean ± SD for triplicate experiments.
8
Cell Cycle Analysis of Melanoma Cells
9
Quantification of dsRNA in Electroporated BHK-21 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BHK-21 cells [American Type Culture Collection (ATCC), Manassas, VA, CCL10] were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gemini, #100-106), MEM nonessential amino acids (Gibco, #11140050), and penicillin/streptomycin/glutamine (Gibco, #10378016). BHK-21 cells were electroporated in strip cuvettes with 3 μg of RNA per 106 cells using SF buffer (Lonza) and 4D-Nucleofector. At 20 hours after electroporation, cells were removed from wells by incubation with trypsin-EDTA solution and washed in PBS containing 5% bovine serum albumin (BSA). The cells were stained for dsRNA using the J2 anti-dsRNA Ab (Scicons, #10010500) conjugated to R-PE using a Lightning-Link R-PE conjugation kit (Innova Biosciences). After staining, cells were evaluated on a ZE5 Cell analyzer (Bio-Rad) and the data were analyzed using FlowJo 10 (Tree Star, Ashland, OR).
10
Huh7 Cell Viability Assay with ALA and DFO
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Huh7 cells (2.1 × 106) were plated on 150-mm tissue culture dishes. After 1 day, the media was switched to include ALA (1 mmol/L) and DFO (100 μmol/L). The cells then were harvested by trypsinization at the indicated time points after initiating the ALA + DFO treatment. Cell viability was estimated using Trypan blue. For fluorescence-activated cell sorter analysis, 106 cells/sample were used. The cells were fixed with ethanol and stained with the nuclear stain FxCycle Far Red (ThermoFisher), and analyzed using a Bio-Rad (Hercules, CA) ZE5 cell analyzer.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!