T2 cells can be loaded with exogenous peptides and β2-microglobulin (β2m) to form specific pHLA on the cell surface. For peptide pulsing, T2 cells in the logarithmic growth phase were cultured at 5 × 105 cells/mL in serum-free IMDM (Gibco, Grand Island, NY, USA) containing 10 μg/mL β2m (Sigma-Aldrich, St. Louis, MO, USA) and either 30 μg/mL irrelevant peptide RMFPNAYL or relevant peptide SLLMWITQC (synthesized by the Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) for 6–8 h at 37 °C with 5% CO2 and 95% humidity. For the cell-binding assay, peptide-pulsed T2 cells were collected and resuspended in 100 μL ice-cold staining buffer (1% BSA in PBS) and then mixed with 1 μg NY-TCRmut (affinity-enhanced NY-TCR). After incubation on ice for 30 min, cells were washed twice with staining buffer, and FITC labelled goat anti-human IgG (H + L) (Beyotime Biotechnology, Shanghai, China) were then added and incubated on ice for 30 min. Finally, cells were examined by flow cytometry (NovoCyteTM, ACEA Biosciences, San Diego, CA, USA) after being washed with staining buffer.
Novocytetm
Manufactured by Agilent Technologies
Sourced in United States
The NovoCyteTM is a flow cytometer designed for cell analysis and sorting. It offers high-performance data acquisition and analysis capabilities. The NovoCyteTM is capable of detecting and measuring multiple parameters of individual cells or particles in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Software, by FlowJo (5 mentions)
Lsrfortessa, by BD (3 mentions)
C6 flow cytometer, by BD (3 mentions)
Red blood cell lysis buffer, by BioLegend (2 mentions)
Mouse igg1kappa isotype control pe, by BioLegend (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Pe anti human cd25, by BioLegend (1 mentions)
Apc anti human cd69, by BioLegend (1 mentions)
Apc anti human cd107a, by BioLegend (1 mentions)
Pe anti human cd8, by BioLegend (1 mentions)
Mouse igg1kappa isotype control apc clone mopc 21, by BioLegend (1 mentions)
Anti human cd3 apc, by BioLegend (1 mentions)
Cytomic fc 500mcl, by Beckman Coulter (1 mentions)
Annexin 5 fitc pi, by MultiSciences Biotech (1 mentions)
Rnase a, by MultiSciences Biotech (1 mentions)
Propidium iodide, by MultiSciences Biotech (1 mentions)
Clodronate liposome, by Liposoma (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
Streptavidin apc, by BioLegend (1 mentions)
19 protocols using novocytetm
1
Peptide Pulsing and T-cell Binding Assay
2
Comprehensive Flow Cytometry Analysis
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All samples were analyzed using a NovoCyteTM (ACEA Biosciences), LSR Fortessa, or C6 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA). The antibodies used included anti-MSLN-biotin (clone MB), Streptavidin-APC, anti-human CCR7-APC (clone 3D12), anti-human CD62L-PE (clone DREG-56), anti-human CD45RA-APC (clone HI100), anti-human CD45RO-PE (clone UCHL1), anti-human TIM3-PE (clone F38-2E2), anti-human LAG3-PerCP/Cy5.5 (clone 11C3C65), anti-human PD-1-APC (clone NAT105), anti-human CD27-PE (clone M-T271), anti-human CD28-APC (clone CD28.2), anti-human CD25-PE (clone BC96), anti-human CD69-APC (clone FN50), anti-human CD107a-APC (clone H4A3), anti-human CD3-APC (clone UCHT1), anti-human CD4-PerCP/Cy5.5 (clone OKT4), anti-human CD8-PE (clone OKT8), mouse IgG2a isotype control-APC (clone RMG2a-62), mouse IgG1kappa isotype control-PE, mouse IgG1kappa isotype control-PErCP/Cy5.5, and mouse IgG1kappa isotype control-APC (clone MOPC-21) (Biolegend, San Diego, CA, USA). All FACS-related staining procedures were performed on ice for 30 min, and cells were then washed with PBS containing 1% FBS before cytometry analysis. PB, spleen (SP), and tumor samples from mouse xenografts were treated with red blood cell lysis buffer (Biolegend), and the cells were stained with the corresponding antibodies.
3
Oxidative Stress Measurement in Caco-2 Cells
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Caco-2 cells were treated as described above. Following this, the cells of each group were collected and stained with DCFH-DA (15 μmol/L) for 20 min. Fluorescence intensity was recorded by flow cytometry (NovoCyte TM, ACEA Biosciences, San Diego, CA, USA) with the excitation of 485 nm and emission of 535 nm. The result was analyzed using NovoExpress software.
4
Oxidative Stress Response in HK-2 Cells
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HK-2 cells were seeded into 12-well plates and incubated with H2O2 (500 μM) and different samples (Cy5-SC and Cy5-C at a concentration of 500 μg/ml) for a predetermined time (2 and 6 hours), referring published papers (14 (link), 47 (link)–52 (link)). After washing with PBS, the cells were harvested and tested by flow cytometry (ACEA NovoCyteTM, ACEA Biosciences, USA). Normal HK-2 cells were also tested as control.
5
Cell Cycle and Apoptosis Analysis
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3 × 105 DLD1 or HCT116 cells were seeded onto 6 well plates, incubated for 24 h at 37 °C, and treated as indicated. For cell cycle analysis, cells were trypsinized, resuspended and fixed with 70% ethanol at −20 °C for at least 1 h. Before analysis, the cells were resuspended in PBS containing 10 mg/mL RNaseA (Multi sciences, China) and 50 mg/mL propidium iodide (PI; Multi sciences, China) for at least 30 min. For apoptosis analysis, the supernatant was transferred to eppendorf tubes and cells were trypsinized and then collected to supernatant and washed twice with cold PBS, followed by staining with Annexin V-FITC/PI (Multi sciences, China) for 15 min. PI- and Annexin V-stained cells were analyzed immediately on a ACEA NovoCyteTM (ACEA Biosciences, USA) or Cytomic FC 500MCL (BECKMAN COULTER, USA).
6
Intratracheal Clodronate Liposomes for AM Depletion
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Mice were injected intratracheal with 50 μl clodronate liposomes (Liposoma) to deplete AMs 2 days before the virus challenge. Control mice received an equal volume of empty control liposomes. AMs depletion was verified by cell flow cytometry using an ACEA NovoCyteTM (ACEA Biosciences).
7
Cell Counting with Novo Cyte Flow Cytometer
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A Novo CyteTM flow cytometer (ACEA Biosciences, Inc., San Diego, CA) was used to determine cell numbers. An aliquot (50 or 100 μL) of each culture was taken and mixed with sheath fluid (950 or 900 μL) containing 0.5% paraformaldehyde, a fixative.
8
Cell Cycle Analysis of hESCs
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Cell cycle analysis of hESCs treated with RO3306 or vehicle was performed using Cell Cycle and Apoptosis Analysis Kit (Beyotime, #C1052). Cells were collected and washed with cold PBS. They were then fixed with pre-cooled 70% ethanol for 30 min at 4°C. After incubated in PI stain solution for 30 min at 37°C, flow cytometry analyses were performed using an ACEA NovoCyteTM (ACEA Biosciences) and ModFit LT (V.4.0.5).
9
Flow Cytometric Analysis of CAR and GL Expression
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To evaluate CAR or GL expression in transduced cells, flow cytometry detections for CD19-, MSLN-CAR NK-92 cells, along with N87 GL, MKN-28 GL, AGS GL, and Huh-7 GL, were performed on a NovoCyteTM (ACEA Biosciences). Transduced CAR NK or GL cells were sorted by a FACS sorter (Beckman Coulter MoFlo XDP, USA). All the data was analyzed by FlowJo software (FlowJo).
10
Mesothelin-Targeted CAR-T Cell Evaluation
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All samples were analyzed using a NovoCyteTM (ACEA Biosciences), LSR Fortessa or C6 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA). The antibodies used for flow cytometry included biotinylated human mesothelin (catalog 296-580) (Acrobiosystems, Beijing, China), streptavidin-APC, and anti-human CD3-APC (clone UCHT1) (BioLegend, San Diego, CA, USA). The transduction efficiency of MSLN CAR-T cells was determined by incubation of 2×105 T cells with 1ug/ml biotinylated human mesothelin at 4°C for 30mins, then washed with PBS containing 1% FBS and incubated with streptavidin-APC for another 30 mins, followed by washing with PBS containing 1% FBS and resuspended with PBS for detection. All the other FACS-related staining procedures were performed on 4°C for 30min, and cells were then washed with PBS containing 1% FBS at 300g before cytometric analysis.
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