For CCK8 analysis, 8000 cells were seeded into each well of a 96-well plate with fresh culture medium. Cell Counting Kit-8 (Solarbio, # CA1210) was used to detect cell viability after 48 h of transfection treatment, and the microplates were incubated at 37 °C. After 4 h, absorbance was recorded at 450 nm using a microplate reader (ThermoFisher) and the relative viability calculated using untreated cells as control (100%). For EdU (5-Ethynyl-2′-deoxyuridine) assay, EdU (Beyotime, # C0071S) was added to the six-well plate and incubated the cells in the logarithmic growth phase for 3 h according to the provided instruction. The cells were washed twice with PBS for 5 min, and then fixed with 4% paraformaldehyde for 30 min. The cells were washed twice with PBS for 5 min each time and infiltrated with 0.3% TritonX-100 in PBS before being stained with the reaction solution. The images were captured with a Leica laser confocal microscope. For the apoptosis assay, cells were incubated staining using Annexin V-PI Apoptosis Detection Kit (Beyotime, #C1065M) according to the provided instruction, followed by flow cytometry analysis (Beckman).
Flow cytometry analysis
Manufactured by Beckman Coulter
Sourced in United States
Flow cytometry is an analytical technique used to identify and quantify specific cell populations within a heterogeneous sample. It involves the measurement of physical and fluorescent characteristics of cells or particles as they pass through a laser beam. The core function of flow cytometry is to provide rapid, quantitative analysis of multiple parameters for individual cells in a heterogeneous population.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (4 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (2 mentions)
Laser confocal microscope, by Leica (1 mentions)
Annexin 5 pi apoptosis detection kit, by Beyotime (1 mentions)
Cell counting kit 8 (cck8), by Solarbio (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Beyotime (1 mentions)
Cd146, by Thermo Fisher Scientific (1 mentions)
Anti cd44 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd24 pe, by Thermo Fisher Scientific (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Annexin 5 fitc, by Sungene Biotech (1 mentions)
Annexin 5, by BioLegend (1 mentions)
7 aad, by BioLegend (1 mentions)
Neural tissue dissociation kit, by Miltenyi Biotec (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
Ms column, by Miltenyi Biotec (1 mentions)
20 protocols using flow cytometry analysis
1
Cell Viability, Proliferation, and Apoptosis Assays
2
Umbelliprenin-Induced Apoptosis in BXPC-3 Cells
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To investigate the extent of umbelliprenin‐induced apoptosis in BXPC‐3 cells, Annexin V/PI staining assay was performed. Briefly, BXPC‐3 cells (1 × 105 cells/well) were treated with different concentrations of umbelliprenin (0, 10, 20, and 40 μg/mL) for 24 h. Then the cells were harvested, washed, and stained with Annexin V‐FITC/PI Apoptosis Detection kit (BD Biosciences). After 20 min incubation, the flow cytometry analysis was performed (Beckman Coulter, Inc.).
3
Flow Cytometry Analysis of HPDLSC Markers
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HPDLSC surface markers were analyzed by flow cytometry. HPDLSCs (P4) were trypsinized and adjusted to 5 × 106 cells/mL, and 1 μg of antibody (CD45, CD146, CD90, and CD73; eBioscience, San Jose, CA, USA) was added to 200 μL of cell suspension. The samples were incubated at 4 °C in the dark for 2 h and centrifuged at 800 r/min for 5 min. The supernatant was discarded, and the cell pellet was washed twice with PBS and resuspended in 200 μL of PBS for flow cytometry analysis (Beckman Coulter, CA, USA).
4
Flow Cytometric Analysis of CD24/CD44 Expression
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Cells exposed to indicated concentration of flubendazole for 72 hr, were trypsinized, washed twice with PBS by centrifugation (1,000 rpm, 5 min), and blocked (0.5% BSA/PBS) for 10 min. Collected cells were then incubated with anti-CD24 PE (eBioscience) and anti-CD44 FITC (eBioscience) or isotype controls antibodies according to manufacturer's instructions for 30 min at 4 °C protected from light. Then, cells were suspended in 1 ml PBS and subjected to flow cytometry analysis (Beckman). Side-scatter and forward-scatter profiles were used to eliminate cell doublets [51 (link)]. The statistical results of three independent experiments were presented.
5
Evaluating Oxidative Stress in NPCs
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ROS levels in NPCs were gauged using Reactive Oxygen Species Assay Kit (Beyotime, China). NPCs were pre-exposed to the complate DMEM medium with 200 μM H2O2 for 6 h before the experiment. Then, the NPCs were categorized into five groups: control (Con), H2O2-treated (H2O2), H2O2 + PEV-treated (PEV), H2O2 + FG-treated (FG), and H2O2 + FG@PEV-treated (FG@PEV) groups. These groups were then incubated for another 24 h. Subsequently, intervertebral disc nucleus pulposus cells were incubated with a 20 mM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime, China) at 37 °C for 20 min. Afterward, the cells underwent three rinses with serum-free medium before proceeding to flow cytometry analysis (Beckman, USA). The fluorescence signal intensity was evaluated by quantifying the oxidative conversion of DCFH-DA. It is important to note that all experiments were conducted in quadruplicate.
6
Cell Viability and Cell Cycle Analysis
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Cells were grown in RPMI-1640 medium containing 10% fetal serum. 1×103 cells were seeded in flat-bottom 96 well plates and incubated at 37°C in 5% CO2. Cell viability was measured using Cell Counting Kit-8 (Dojindo Laboratories) at day1,day3 and day5. For cell cycle analysis, cells were washed and fixed with ice-cold 75% (v/v) ethanol at −20°C for 2 h, then stained with PI at the concentration of 50 µg/mL in the presence of RNase A (100 µg/mL). DNA content was analyzed by Flow cytometry analysis (Beckman Coulter, USA).
7
Apoptosis Detection via Annexin V-FITC/PI
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Common-used Annexin V-FITC/PI apoptosis detection kit (Becton-Dickinson, Franklin Lakes, NJ, USA) was utilized to examine the percentage of apoptotic cells as described previously [18 (link)]. After stimulation with 30 μM of Swainsonine for 12 h, U251 and LN444 cells were baptized in PBS, and stained with 5 μL PI/FITC-Annexin V for 30 min in the absence of light at ambient temperature. After staining, cell apoptosis was straightway evaluated by utilizing flow cytometry analysis (Beckman Coulter, Fullerton, CA, USA).
8
Cell Cycle, Apoptosis, and ROS Analysis
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Cells were collected and washed three times with PBS. For the cell cycle analysis, cells were re-suspended in 1 mL DNA staining solution. 10 μL propidium iodide (PI) solution (MultiSciences Biotech, China) was added. The cells were incubated in darkness for at least 30 min. For the apoptosis analysis, cells were re-suspended in 100 μL 1× Annexin V binding buffer, and then, we sequentially added 5 μL Annexin V-FITC and 5 μL PI solution (Sungene Biotech, China). Another 400 μL 1× Annexin V binding buffer was added after 30 min of incubation. A parameters regulation was performed at each independent experiment according to the user’s instructions. For the ROS analysis, cells were re-suspended in 1 mL FBS-free medium, and 1 μL DCFH-DA (Sigma, D6883) was added. After 30 min of incubation, cells were centrifuged and re-suspended in a 1 mL FBS-free medium. Each sample was analyzed using flow cytometry analysis (Beckman, USA).
9
miR-708 and NRAS Mediated Apoptosis
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Cells transfected with miR-708 or siNRAS were treated with 200 μM hydrogen peroxide (H2O2) (Showa Denko, Japan) to induce apoptosis for 24 hr after 48 hr transfection. Cells were then collected and double stained with 80 ng/mL Annexin V (BioLegend, 640920) and 5 μg/mL 7-AAD (BioLegend, 420403) in calcium-rich binding buffer (10mM pH 7.8 HEPES, 140mM NaCl, 2.5mM CaCl2) for 15 minutes. The stained cells were then subjected to flow cytometry analysis (Beckman Coulter) and the raw data was analyzed by using CytExpert 2.0. Data are means ± SD from 3 independent experiments and presented as fold changes relative to the mock control.
10
Microglia Isolation from Mouse Brain
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Brain tissue from HICT and SED healthy or pre-onset EAE mice were dissociated to single-cell suspension, using the Neural Tissue Dissociation Kit (Miltenyi Biotec). Myelin was removed using Percoll (GE Healthcare) followed by microglia isolation using CD11b microbeads and MS columns (Miltenyi Biotec) according to manufacturer instructions. The degree of microglia enrichment was assessed by CD11b (BD Bioscience, M1/70) staining and flow cytometry analysis (Beckman Coulter). In all experiments at least 85% of isolated cells expressed CD11b.
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