flow cytometry was performed to detect reactive oxygen species (ROS) formation, apoptosis, and mitochondrial membrane potential (MMP) in H9c2 cells. For the ROS generation assay, 1 × 106 H9c2 cells in each group were resuspended in 1 mL of diluted DCFH-DA and maintained at 37°C for 20 min. Thereafter, the cells were treated with 500 μL phosphate-buffered saline (PBS) and analyzed with flow cytometry (ACEA Biosciences, San Diego, CA, USA). For the apoptosis assay, 1 × 106 H9c2 cells in each group were centrifuged at 400 × g at 4°C for 5 min, resuspended in 200 μL PBS, and stained for 30 min with 10 μL annexin V-fluorescein isothiocyanate (FITC) and 10 μL propidium iodide (PI) in the dark at 4°C. After 300 μL PBS was added, the cells were analyzed with flow cytometry (ACEA Biosciences). For the MMP assay, 1 × 106 H9c2 cells in each group were resuspended in 500 μL DMEM and cultured for 20 min with JC-1 solution (Beyotime) at 37°C. Thereafter, the cells were centrifuged at 400 × g at 4°C for 3 min and resuspended in 1 mL of JC-1 solution. The cells were then centrifuged at 400 × g at 4°C for 3 min, resuspended in 400 μL of JC-1 solution, and analyzed with flow cytometry (ACEA Biosciences).
Flow cytometry
Manufactured by Agilent Technologies
Sourced in United States, China
Flow cytometry is an analytical technique used to measure and analyze multiple physical characteristics of particles, such as cells, within a fluid as they pass through a beam of light. The core function of flow cytometry is to provide rapid quantitative and qualitative analysis of particles in a heterogeneous mixture.
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Lab products found in correlation
Novoexpress software, by Agilent Technologies (3 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Dojindo Laboratories (2 mentions)
Novoexpress 1, by Agilent Technologies (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (2 mentions)
Annexin 5 fitc, by Solarbio (2 mentions)
Annexin 5 fitc apoptosis kit, by Solarbio (2 mentions)
Jc 1 solution, by Beyotime (1 mentions)
Annexin 5 fitc apoptosis assay kit, by Keygen Biotech (1 mentions)
Rnase a, by Merck Group (1 mentions)
Annexin 5 fitc propidium iodide apoptosis detection kit, by Vazyme (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Annexin 7aad pe apoptosis detection kit, by BD (1 mentions)
Flow cytometry, by Beckman Coulter (1 mentions)
Annexin 5 apc propidium iodide apoptosis detection kit, by Keygen Biotech (1 mentions)
Jc 1 staining assay kit, by Beyotime (1 mentions)
Atp bioluminescence assay kit, by Beyotime (1 mentions)
Jc 1 assay kit, by Beyotime (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Apoptosis detection kit, by Vazyme (1 mentions)
Ctb fitc, by Merck Group (1 mentions)
61 protocols using flow cytometry
1
Evaluating Cellular Stress Responses
2
Cellular Uptake of Functionalized Polymer Nanomaterials
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To investigate the cellular uptake of PM@RM-T7 into 4T1 cancer cells, Met was replaced with the fluorescent molecule Ce6 to yield PC@RM-T7 as a surrogate. 4T1 cells seeded on six-well microplates (4 × 104 cells per well) were cultured overnight and then incubated with PC@RM and PC@RM-M2 (at the equivalent concentration of mPDA = 200 μg/mL) for 6 h. After being washed with PBS, the cells were digested by trypsin and collected for flow cytometry analysis (Agilent, Santa Clara, CA, USA). Furthermore, a laser scanning confocal microscope (CLSM, Zeiss, Oberkochen, German) was employed to evaluate the cellular uptake of PC@RM-T7 by 4T1 cells qualitatively.
To investigate the phagocytosis of PR@RM-M2 into M2 type RAW264.7 cells. RAW264.7 cells were polarized into M2 type by IL-4 (20 ng/mL) and then incubated with PC@RM, PC@RM-T7, and PC@RM-M2 (at the equivalent concentration of mPDA = 200 μg/mL) for 24 h. flow cytometry (Agilent) and CSLM (Zeiss) were utilized to quantitatively and qualitatively analyze the cellular uptake.
To investigate the phagocytosis of PR@RM-M2 into M2 type RAW264.7 cells. RAW264.7 cells were polarized into M2 type by IL-4 (20 ng/mL) and then incubated with PC@RM, PC@RM-T7, and PC@RM-M2 (at the equivalent concentration of mPDA = 200 μg/mL) for 24 h. flow cytometry (Agilent) and CSLM (Zeiss) were utilized to quantitatively and qualitatively analyze the cellular uptake.
3
Investigating Tumor Immune Modulation
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Mice with 4T1 tumors were randomly split into 6 treatment groups (n = 3): saline, HTC, MARS, RT, HTC+RT, or MARS+RT. RT was administered 4 h after the second intravenous injection at a dose of 4 Gy. After completing the entire treatment, we euthanized them and harvested their tumors, spleens, and tumor-draining lymph nodes (TDLNs) to make a single-cell suspension. The cell suspension was stained with anti-mouse CD11c, anti-mouse CD86, and anti-mouse CD80 antibodies for flow cytometry (Agilent) to investigate DCs at different maturation levels. The tumor single-cell suspensions were labeled with fluorophore-conjugated antibodies (anti-mouse CD3, anti-mouse CD4, anti-mouse CD25, and anti-mouse FoxP3) and evaluated by flow cytometry (Agilent) to identify Tregs. The TDLNs cell suspensions were stained with anti-mouse CD3, anti-mouse CD8a, and anti-mouse CD4 antibodies and then identified using flow cytometry (Agilent) to assess T-cell activation. The cytokine levels of interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) in the serum were analyzed using corresponding ELISA kits according to the reagent instructions.
4
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, cells treated with or without PDS were harvested and fixed in ice-cold 70% ethanol, stained with 1x PBS based propidium iodide solution (50 μg/ml PI, 100 μg/ml RNase A, 0.1% sodium citrate, 0.1% Triton X-100), and analyzed by flow cytometry (Agilent, USA).
For Annexin V staining, cells treated with or without PDS were harvested and stained with Annexin V-FITC and PI solution, and analyzed by flow cytometry (Agilent).
For Annexin V staining, cells treated with or without PDS were harvested and stained with Annexin V-FITC and PI solution, and analyzed by flow cytometry (Agilent).
5
Exosome Regulation of HemEC Apoptosis and Cell Cycle
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HemECs were cultured in 6-well plates and then cultured with exosomes derived from HemSCs or HUVECs when the density reached 105 cells/well. After 48 h, cells were harvested and centrifuged at 1,000 r at 4 °C, and resuspended again in 1× binding buffer. Annexin V-FITC apoptosis assay kit (Nanjing KeyGen Biotech. Co. Ltd., China) was used to analyze cell apoptosis within 15 min in the dark. Flow cytometry (Agilent Technology, USA) was utilized to determine the apoptotic rate.
HemECs were cultured in 6-well plates (2×105 cells/well) and then cultured with exosomes derived from HemSCs or HUVECs. After 48 h, the cells were digested and collected. After being fixed in 75% ethanol for 2 h, the cells were treated with RNase A (Sigma Aldrich, USA) and stained in 500 mL PI (Sigma Aldrich, USA). Flow cytometry (Agilent Technology, USA) was employed to analyze the cell distribution in each phase, and cells in G0/G1, S, and G2/M phases in each group were counted.
HemECs were cultured in 6-well plates (2×105 cells/well) and then cultured with exosomes derived from HemSCs or HUVECs. After 48 h, the cells were digested and collected. After being fixed in 75% ethanol for 2 h, the cells were treated with RNase A (Sigma Aldrich, USA) and stained in 500 mL PI (Sigma Aldrich, USA). Flow cytometry (Agilent Technology, USA) was employed to analyze the cell distribution in each phase, and cells in G0/G1, S, and G2/M phases in each group were counted.
6
Cellular Uptake of Functionalized Polymer Nanomaterials
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To investigate the cellular uptake of PM@RM-T7 into 4T1 cancer cells, Met was replaced with the fluorescent molecule Ce6 to yield PC@RM-T7 as a surrogate. 4T1 cells seeded on six-well microplates (4 × 104 cells per well) were cultured overnight and then incubated with PC@RM and PC@RM-M2 (at the equivalent concentration of mPDA = 200 μg/mL) for 6 h. After being washed with PBS, the cells were digested by trypsin and collected for flow cytometry analysis (Agilent, Santa Clara, CA, USA). Furthermore, a laser scanning confocal microscope (CLSM, Zeiss, Oberkochen, German) was employed to evaluate the cellular uptake of PC@RM-T7 by 4T1 cells qualitatively.
To investigate the phagocytosis of PR@RM-M2 into M2 type RAW264.7 cells. RAW264.7 cells were polarized into M2 type by IL-4 (20 ng/mL) and then incubated with PC@RM, PC@RM-T7, and PC@RM-M2 (at the equivalent concentration of mPDA = 200 μg/mL) for 24 h. flow cytometry (Agilent) and CSLM (Zeiss) were utilized to quantitatively and qualitatively analyze the cellular uptake.
To investigate the phagocytosis of PR@RM-M2 into M2 type RAW264.7 cells. RAW264.7 cells were polarized into M2 type by IL-4 (20 ng/mL) and then incubated with PC@RM, PC@RM-T7, and PC@RM-M2 (at the equivalent concentration of mPDA = 200 μg/mL) for 24 h. flow cytometry (Agilent) and CSLM (Zeiss) were utilized to quantitatively and qualitatively analyze the cellular uptake.
7
Apoptosis Detection in HCT Cell Lines
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HCT-8 and HCT-116 cells, transfected with plasmids as above described, were cultured in 6-well plates at a density of 5×105 cells per well. After 48 h, cells were stained using Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (VAZYME, Catalog No. A211-01, Nanjing, Jiangsu, China) as previously described.21 (link) Flow cytometry (ACEA Biosciences, Inc., San Diego, CA, USA) was then used for detection.
8
Annexin V/JC-1 Apoptosis Assay Protocol
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AGS and N87 were incubated in a 6‐well cell culture plate (1×105 cells per well) and treated by different concentrations for 2days. After treatment, the cells were then washed by 1x binding buffer, 5μL of Annexin V/FITC was added, and finally incubated for 5minutes. ACEA NovoCyteTM flow cytometry was used to analyze the stained cells. For JC‐1 staining, cells were seeded into 6‐well plates (4×103 cells per well) and cultured for 24hours. The cells were collected and washed with PBS, then incubated at 37°C with JC‐1 for 15minutes. After the dye was washed away, cells were analyzed immediately using flow cytometry (ACEA biosciences).
9
Quantifying Intracellular ROS and Hydroxyl Radicals
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The fluorescence detection probes carboxy-2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) with excitation (Ex) and emission (Em) wavelengths of 495 and 525 nm, respectively, and HPF (Ex/Em = 490/515 nm) were applied to respectively evaluate the intracellular ROS and ·OH level in 4T1 tumor cells, respectively. In brief, 4T1 cells were treated with FGP or FHP (with an equivalent Fe concentration of 180 μg/mL) for 2 h. The cells were rinsed and stained with carboxy-H2DCFDA (20 μmol/L in serum-free medium) and HPF (10 μmol/L) at 37 °C for 30 min. The cells were then fixed in 4% formaldehyde polymer and labeled with 4ʹ,6-diamidino-2-phenylindole (DAPI, 1 μg/mL), and washed twice with PBS. Finally, the fluorescence images of cells were captured by a fluorescence microscopy and the mean fluorescence was calculated by Image J software (NIH, Bethesda, MD, USA). The ROS generation was also similarly evaluated by flow cytometry (ACEA Biosciences) using 4T1 cells seeded in six-well plates (1 × 106 cells) per well, centrifuged when required, and suspended in PBS for analysis.
10
Annexin V-FITC/PI Apoptosis Assay
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An annexin V-FITC/PI apoptosis detection kit (AD10, Dojindo Laboratories, Shanghai, China) was used for apoptotic cell determination with a flow cytometry (ACEA Biosciences, Inc.) following the instruction. The apoptotic cell percentage was defined as the sum of the early and late apoptotic cell percentages.
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