Antibody-dependent cellular phagocytosis was performed as previously described 63 (link) with slight modifications. Briefly, goat-anti human IgG F(ab’)2 (Invitrogen) was covalently coupled to yellow-green carboxylate beads (Thermofisher). Antibodies were diluted in culture medium to a starting concentration of 133 nM and serially diluted 4-fold 7 times. Diluted mAbs were incubated with anti-human IgG beads for 2 hours at 37°C to form immune complexes. THP-1 (ATCC) cells (25,000/well) were added to the immune complexes and incubated at 37°C for 4 hours. Cells were washed 2x with cold 1x PBS prior to being fixed with 4% paraformaldehyde. The cells were analyzed on a NovoCyte Advanteon flow cytometer (Agilent) (Figure S2C ). A phagocytosis score was calculated as the (percentage of FITC+ cells) x (the geometric mean fluorescence intensity (gMFI) of the FITC+ cells)/100,000. Buffer only wells were used as negative controls and the assay was performed in technical replicate with two biological replicates.
Novocyte advanteon flow cytometer
Manufactured by Agilent Technologies
Sourced in United States
The NovoCyte Advanteon is a flow cytometer designed for advanced cell analysis. It features a compact, benchtop design and provides high-performance capabilities for researchers and clinicians. The core function of the NovoCyte Advanteon is to detect and analyze individual cells or particles suspended in a fluid stream.
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Lab products found in correlation
Goat anti human igg f ab 2, by Thermo Fisher Scientific (2 mentions)
Yellow green carboxylate beads, by Thermo Fisher Scientific (2 mentions)
Cytometric bead array flex set beads, by BD (2 mentions)
Bck fc488 50, by Merck Group (2 mentions)
Liberase, by Merck Group (1 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
Rock inhibitor, by Merck Group (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Solarbio (1 mentions)
Fluorescence microscopy, by Leica (1 mentions)
Mitotracker red cm h2xros, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Dojindo Laboratories (1 mentions)
Ros detection reagent, by Thermo Fisher Scientific (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
13 protocols using novocyte advanteon flow cytometer
1
Antibody-Dependent Cellular Phagocytosis Assay
2
Phagocytosis of Aeromonas hydrophila by Largemouth Bass Leukocytes
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A. hydrophila was cultured in LB medium at 37 °C and collected in the logarithmic growth phase. The collected bacteria were washed three times with 0.1 mM sodium bicarbonate solution and incubated with 4% paraformaldehyde at 25 °C for 2 h to inactivate the bacteria. The inactivated A. hydrophila were labeled with 0.1 mg/mL fluorescein isothiocyanate (FITC) at 25 °C for 2 h, washed with PBS five times to remove excess FITC, and finally resuspended to 1.0 × 108 CFU/mL for use.
The leukocytes were isolated from largemouth bass by Percoll density gradient. The cells were resuspended with 1640 medium and the cell concentration was adjusted to 1.0 × 106 CFU/mL. Then, 500 μL of leukocyte suspension was incubated with 5 μL anti-rCRD antibody or mouse negative serum for 1 h at 25 °C, respectively. Then, 50 μL of FITC-labeled A. hydrophila was added and the mixtures were incubated at 25 °C for 30 min. Following incubation, the cell mixtures were laid over 3 mL PBS containing 3% BSA and 4.5% D-glucose and centrifuged at 100 g at 4 °C for 10 min to remove unengulfed A. hydrophila. Finally, the leukocytes were washed three times with PBS and detected using a NovoCyte Advanteon Flow Cytometer (Agilent, Santa Clara, CA, USA).
The leukocytes were isolated from largemouth bass by Percoll density gradient. The cells were resuspended with 1640 medium and the cell concentration was adjusted to 1.0 × 106 CFU/mL. Then, 500 μL of leukocyte suspension was incubated with 5 μL anti-rCRD antibody or mouse negative serum for 1 h at 25 °C, respectively. Then, 50 μL of FITC-labeled A. hydrophila was added and the mixtures were incubated at 25 °C for 30 min. Following incubation, the cell mixtures were laid over 3 mL PBS containing 3% BSA and 4.5% D-glucose and centrifuged at 100 g at 4 °C for 10 min to remove unengulfed A. hydrophila. Finally, the leukocytes were washed three times with PBS and detected using a NovoCyte Advanteon Flow Cytometer (Agilent, Santa Clara, CA, USA).
3
Antibody-Dependent Cellular Phagocytosis Assay
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Antibody-dependent cellular phagocytosis was performed as previously described78 (link) with slight modifications. Briefly, goat-anti human IgG F(ab’)2 (Invitrogen) was covalently coupled to yellow-green carboxylate beads (Thermofisher). Antibodies were diluted in culture medium to a starting concentration of 133 nM and serially diluted 4-fold 7 times. Diluted mAbs were incubated with anti-human IgG beads for 2 h at 37°C to form immune complexes. THP-1 (ATCC) cells (25,000/well) were added to the immune complexes and incubated at 37°C for 4 h. Cells were washed 2x with cold 1x PBS prior to being fixed with 4% paraformaldehyde. The cells were analyzed on a NovoCyte Advanteon flow cytometer (Agilent) (Figure S2 C). A phagocytosis score was calculated as the (percentage of FITC+ cells) x (the geometric mean fluorescence intensity (gMFI) of the FITC+ cells)/100,000. Buffer only wells were used as negative controls and the assay was performed in technical replicate with two biological replicates.
4
Tumor Xenograft Cell Isolation and ROS Measurement
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The tumor xenograft to be tested was first diced into small pieces of approximately 1 mm3 with surgical blades and dissociated into a single-cell suspension by the gentleMACS dissociator with a series of 3 human tumor dissociation programs (h-tumor_01.01, h-tumor_02.01, and h-tumor_03.01) in the presence of DMEM/F12 culture medium with 20 mmol/L ROCK inhibitor (Sigma), 2.5 mg/mL liberase (Sigma), and 100 mg/mL DNAse I (Roche). Each step was performed twice and a 5-minute incubation at 37ºC was introduced between the 3 different programs. The cell suspension was centrifuged at 900 rpm for 4 minutes, and the cell pellets were washed with DMEM/F12 twice to remove the red blood cells and unwanted cell debris. The number of viable cells was evaluated with Trypan blue staining using a Countess II automated cell counter (Life Technologies). To evaluate the intracellular ROS level, cell pellets consisting of 1 × 105 live cells were resuspended in 1 mL ice-cold PBS with 2 umol/L CM-H2DCFDA (Invitrogen). The fluorescence signal was acquired by the Novocyte Advanteon Flow Cytometer (Agilent) and analyzed with FlowJo v10.7 (BD).
5
NPC Viability and Apoptosis Assay
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Cell viability of N-NPC, Y-NPC and O-NPC was detected using CCK8 kit (Dojindo, Janpan). Briefly, the concentration of each NPC was adjusted to 5 × 105/ml and inoculated in 96-well plates. A blank control well was set up, and 3 replicate wells were set up for the experimental group. After each NPC was incubated for 0, 12, 24, 48 and 72 h, 10 µl CCK8 solution was added to each well and incubated in the incubator for 4 h. Subsequently, the optical density (OD) of each well was measured at 450 nm using a ELX800 microplate reader (Biotech, USA).
Apoptosis ratios of N-NPC and Y-NPC were detected using ANNEXIN V- FITC/PI Apoptosis Detection Kit (Solarbio). According to the instructions, 5 µl FITC and 10 µl PI were added to N-NPC and Y-NPC after trypsin digestion and incubated for 15 and 5 min, respectively, in the dark. Subsequently, the proportion of cell apoptosis in each group was detected using a Novocyte Advanteon flow cytometer (Agilent, USA).
Apoptosis ratios of N-NPC and Y-NPC were detected using ANNEXIN V- FITC/PI Apoptosis Detection Kit (Solarbio). According to the instructions, 5 µl FITC and 10 µl PI were added to N-NPC and Y-NPC after trypsin digestion and incubated for 15 and 5 min, respectively, in the dark. Subsequently, the proportion of cell apoptosis in each group was detected using a Novocyte Advanteon flow cytometer (Agilent, USA).
6
Glycoconjugate-Induced Cytokine Production
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The animals (n = 10 BALB/c mice per group and n = 10 ICR mice per group) received two intraperitoneal injections (100 μg glycoconjugate no. 10 in 200 μL PBS) at 21-day intervals. On day 26, spleens were aseptically removed from all animals in all groups, and splenocytes were suspended in a DMEM medium with 10% fetal bovine serum and placed into the wells of 24-well culture plates (2 × 106 cells/well). In parallel, splenocytes were also obtained from intact mice (n = 10 mice per group) without prior immunization with glycoconjugate. Various amounts of K9 CPS solution (1, 10, and 100 μg) were added to the cells in 3 independent repeats, and these served as the sources of the carbohydrate moiety during glycoconjugate synthesis. The plates were incubated for 48 h, after which the contents of IL-4, IL-10, IL-17A, γ-IFN, and TNF-α were analyzed using BD Cytometric Bead Array flex set beads (mouse IL-4 [cat. no 558298], mouse IL-10 [cat. no 558300], mouse IL-17A [cat. no 560283], mouse γ-IFN [cat. no 558296], and mouse TNF-α [Cat. No 558299]) according to the manufacturer's instructions. The measurements were performed using a NovoCyte Advanteon flow cytometer (Agilent, USA).
7
Mitochondrial Mass Quantification
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Mitochondrial mass was assessed with MitoTracker™ Red CM‐H2Xros (Invitrogen) according to the manufacturer's protocol. Briefly, cells were incubated with MitoTracker for 30 min at 37°C followed by fluorescence microscopy (Leica) or analysis using a NovoCyte Advanteon flow cytometer (Agilent).
8
Apoptosis Analysis of 5-FU and CAP
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After treated with 5-FU or CAP at the indicated administration, approximately 1 × 106 cells per group were collected and centrifuged at 3, 000 rpm for 5 min and washed with PBS. Subsequently, cells were stained using an Annexin V FITC/PI staining Apoptosis Detection kit according to the manufacturer’s instructions. Briefly, annexin V-FITC and PI were added to cell suspension in buffer Binding. The cells were incubated at room temperature and kept away from light for 20–30 min, and then the cells were resuspended. The percentage of apoptotic cells for each sample was analyzed by Novocyte Advanteon flow cytometer (Agilent, USA) and NovoExpress software 14.1.
9
Click-IT EdU Proliferation Assay
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The Click-IT EdU protocol was used according to manufacturer instructions (Sigma-Aldrich #BCK-FC488-50). Briefly, MEFs were seeded at 50,000 cells per well in 6-well plates in DMEM supplemented with 10% FBS and 1% P/S, and allowed to attach overnight. The next day, cells were serum-starved for 5 h prior to compound addition for 24 h in fresh serum-free DMEM. Cells were then pulsed for 3 h with 10 μM EdU, followed by collection by trypsinization and fixation with 3.7% FA in PBS for 15 min in the dark, washed in 3% BSA and permeabilized in 1x saponin-based permeabilization buffer for 20 min in the dark. EdU was then detected using the FAM-azide assay cocktail for 30 min in the dark. Cells were washed twice in 1x saponin-based permeabilization buffer followed by analysis with flow cytometer (Novocyte Advanteon flow cytometer, Agilent). Gating strategy for flow cytometry is shown in Supplementary Fig. 2 .
10
Mitochondrial ROS and Apoptosis Assay
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Mitochondria ROS levels were measured using the MitoSOX™ Red Mitochondrial Superoxide Indicator (Invitrogen) according to the manufacturer's protocol. Cellular ROS levels were measured using ROS Detection Reagents (Thermo Fisher) according to the manufacturer's protocol. Apoptosis assay was performed with an Annexin V‐FITC Apoptosis Detection Kit (Dojindo) according to the manufacturer's instructions. Stained cells were analyzed on a NovoCyte Advanteon flow cytometer (Agilent).
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