BV-2 cells in the culture insert were digested using trypsin and then washed and re-suspended in cold PBS. The cells were fixed with Fix/Perm working solution (Transcription Factor Buffer Set, #562574, BD Pharmingen, CA, USA) for 1 h on ice. The cells were incubated with PE-conjugated CD16/32 antibody (1:200, #561727, BD Biosciences) and Alexa Fluor 647-conjugated CD206 antibody (1:200, #56250, BD Biosciences) for 45 min. The isotype of the CD16/32 antibody or CD206 antibody was used as the negative control. Cells were gated based on FSC/SSC and analyzed using the excitation and emission maximum of approximately 496 and 576 nm (PE-conjugated CD16/32) or 653 and 669 nm (Alexa Fluor 647-conjugated CD206) by Novocyte flow cytometry (ACEA Biosciences, CA, USA).
Novocyte flow cytometry
Manufactured by Agilent Technologies
Sourced in United States, China
The NovoCyte flow cytometry system is a high-performance analytical instrument used for the detection and analysis of cells and particles in a liquid sample. It provides quantitative and qualitative data on various cellular parameters, such as size, granularity, and fluorescence intensity.
Automatically generated - may contain errors
Lab products found in correlation
Novoexpress software, by Agilent Technologies (2 mentions)
Transcription factor buffer set, by BD (1 mentions)
Cytochalasin d cytod, by Merck Group (1 mentions)
Novoexpress 1, by Agilent Technologies (1 mentions)
Apoptosis detection kit, by Vazyme (1 mentions)
A211 01, by Vazyme (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Propidium iodide, by BD (1 mentions)
Ifn β, by BioLegend (1 mentions)
Annexin 5 fluorescein isothiocyanate pi apoptosis detection kit, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Rnase a, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cd105, by BD (1 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (1 mentions)
Live dead baclight bacterial viability and counting kit, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Annexin 5 fitc propidium iodide apoptosis detection kit, by Keygen Biotech (1 mentions)
32 protocols using novocyte flow cytometry
1
Flow Cytometric Analysis of BV-2 Cell Markers
2
Annexin V-FITC/PI Apoptosis Detection
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Annexin V-FITC/PI Cell Apoptosis Detection Kit (AP101-100 kit, MULTI SCIENCES, China) is used to detect cell apoptosis. Through NovoCyte™ Flow cytometry (NovoCell 2060R, ACEA Biosciences Inc., USA) detects apoptosis at a wavelength of 488 nm [30 (link)].
3
Phagocytosis Assay with pHrodo E. coli
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The pH-sensitive green fluorophore-tagged Escherichia coli (E. coli) bioparticles (pHrodo Green E. coli BioParticles Conguates, # P35366, Invitrogen Corporation, Frederick, MD, USA) were used to measure the phagocytosis activity. The E. coli bioparticles are nonfluorescent at neutral pH. When the microglia engulf the bioparticle, phagocytic cargo presents early phagosomes of higher pH, which sequentially mature into late phagosomes, becoming increasingly acidic and finally fusing with the lysosomes for degradation and clearance. Briefly, BV2 microglial cells (5 × 105/well) were plated in 6-well plates and cultured for 24 to 72 h. A total of 100 μg of pHrodo Green Escherichia coli (E. coli) BioParticles Conguates were added per condition and incubated with BV2 cells for 30 min at 37 °C. Phagocytosis was inhibited with 10 μM cytochalasin D (Cyto. D; #SI-C8273, Sigma-Aldrich), which was added 30 min before the addition of PHrodo E. coli bioparticles as a negative control. Cells were gated based on forward scatter (FSC)/side scatter (SSC) properties by Novocyte flow cytometry (ACEA Biosciences, CA, USA). Then adjusted threshold to eliminate debris, and the percentage of the pHrodo + cell was determined.
4
Apoptosis Assay for Primary Cells and Cell Lines
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For primary cells derived from SD rats, single cell suspensions were prepared as mentioned in the “Isolation of lung type II pneumocytes cells” section. For cell lines, A549 cells were digested by trypsin without EDTA. The cell concentration was adjusted to 3 × 10 [5 (link)]/mL and stained with a 100ul binding solution containing 5ul annexin V-fluorescein isothiocyanate and 5ul PI solution in a 100ul binding solution. Apoptosis was assayed using a Novo Cyte flow cytometry (Acea Biosciences, San Diego, CA, USA) .
5
Apoptosis analysis of hFOB1.19 cells co-cultured with hucMSCs or exosomes
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The hFOB1.19 cells co-cultured with hucMSCs or hucMSC-exosomes for 48 h were collected by centrifugation at 500 × g for 5 min, and stained for 20 min using an apoptosis detection kit (catalog number: A211-01, Vazyme, Nanjing, Jiangsu, China) according to the merchant instructions. Then, NovoCyte flow cytometry (ACEA, San Diego, CA, USA) and associated software (NovoExpress 1.4.1, ACEA) were used to analyze the apoptosis ratio.
6
Annexin V-PI Apoptosis Assay
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Cell apoptosis was investigated by dual staining with annexin V-FITC and propidium iodide (BD Pharmingen, NJ, USA). PC12 cells were plated at a density of 1 × 105 per well in 6-well plates and cultured at 37°C for 48 hours. Trypsin was used to dissociate cells from the dishes in which differentiated PC12 cells were being cultured. Cells were centrifuged at 1000 rpm for 10 minutes. Supernatant was removed, and the cells were washed twice with PBS buffer. The cells were suspended in 400 μl binding buffer supplemented with 5 μl annexin V. The mixture was incubated at room temperature in the dark for 10 minutes. PI solution (5 μl) was added, and the mixture was incubated for 15 minutes in the dark at room temperature. Cell apoptosis percentage was determined using NovoCyte flow cytometry(ACEA, USA).
7
Innate Immune Cell Mobilization by Adjuvants
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C57BL/6 female mice (n=4 per group) were administered either with 2 μg of ovalbumin (OVA; InvivoGen) alone or formulated with 10 μg of NVT or10 μg of poly(I:C) as an adjuvant via the intramuscular route. At 0, 6 or 24 h, the draining iLN was harvested to dissociate into a single cell suspension. The cells were labeled with a fixable viability dye to distinguish live cells from dead cells followed by surface staining for macrophage (CD11b+F4/80+), neutrophil (CD11b+Ly6G+), and DCs (CD11chiMHC-IIhi) (Supplementary Figure 1
8
Mitomycin C-Induced Apoptosis and Cell Cycle
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The third generation of endometrial stromal cells or the eighth of uterine fibroblast were selected for apoptosis and cell cycle assays. The cells were seeded in 96-well plates (1×103/well) for 24 hours, and then cultured with 4 mL different concentrations (0, 5, 10 ug/mL) of mitomycin C for 48 hours. After adding 300 uL of 1×Binding Buffer to each tube of cells, the apoptotic cells were marked with 5 uL of Annexin V-FITC and incubated for 15 minutes at room temperature in the dark. Then 5 uL of propidium iodide (PI) was then added and incubated for 5 min at room temperature in the dark. Finally, after adding 200 uL of 1×Binding Buffer, apoptotic cells were analyzed by NovoCyte flow cytometry (ACEA, China). For cell cycle, the cells were only stained with PI and analyzed on the flow cytometry.
9
Mitochondrial Membrane Potential Assay
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Briefly, 100 μL of a JC‐1 working solution was added to cells and incubated in the dark for 30 min at 37°C. Following incubation, cells were washed once and resuspended using 400‐μL staining buffer for analysis via the Novocyte flow cytometry (ACEA Biosciences). A total of 10,000 cells were measured using the FL‐1 channel (525 nm), and results were expressed as a percentage.
10
Cell cycle analysis by PI staining
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Cells were harvested and fixed with 75% ethanol at 4°C overnight. For the analysis, PI staining solution (50 mg/mL PI and 100 mg/mL ribonuclease A) was added to the cells and incubated for 30 minutes in darkness at 37°C. The cells were analyzed using NovoCyte flow cytometry (ACEA Biosciences). Three independent experiments were conducted.
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