The proliferation capacity of the indicated hAMSCs was detected by using the Cell Counting Kit-8 (CCK-8, Japan) and Ki-67 staining (BioLegend, USA)-based FCM assay. Briefly, hAMSCs were incubated with the CCK-8 reagents at 37 °C for 2 h. The absorbance at 450 nm was read by microplate reader (Thermo Fisher, USA).
Ki 67 staining
Manufactured by BioLegend
Sourced in Japan, United States
Ki-67 is a nuclear protein that is associated with cellular proliferation. It is commonly used as a marker for cell division and is often used in the assessment of cancer cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Mouse igg2b control, by BioLegend (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Xtt assay, by Sartorius (1 mentions)
Stat3 inhibitor s3i 201, by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
1 methyl d tryptophan 1 mt, by Merck Group (1 mentions)
Antihuman pd l1 neutralizing antibody, by BioLegend (1 mentions)
4 protocols using ki 67 staining
1
Proliferation Capacity of hAMSCs
2
Modulation of NK Cell and T Cell Proliferation
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Autologous NK cells labeled with CTDR were cultured alone or co-cultured with macrophages with the indicated treatments (2:1) in complete medium (RPMI-1640 supplemented with 10% FBS) and stimulated with 100 U/mL IL-2 and 50 U/mL IL-15 (Cat# 200-15; PeproTech) for 4 days. The proliferation rate was then evaluated by flow cytometry for Ki-67 staining (Cat# 350503; BioLegend). In some experiments, 5 μg/mL anti-human B7-H4 neutralizing Ab (eBioscience) or 5 μg/mL mouse IgG2b control (Cat# 400301; BioLegend) was added to the co-culture.
For the T cell proliferation assay, autologous CD8+ T cells were labeled with 0.5 μM CFSE (Cat# C34554; Thermo Fisher Scientific) for 15 min at room temperature and incubated with mature dendritic cells (DCs; 5:1) and the indicated labeled macrophages (2:1) in RPMI-1640 medium supplemented with 5 μg/mL IL-12, 25 mM HEPES, 4 mM L-glutamine, 25 μM 2-mercaptoethanol, and 10% FBS. Proliferation of CD8+ T cells was measured by CFSE staining and flow cytometry after 4 days. In some experiments, 5 μg/mL anti-human B7-H4 neutralizing Ab or 5 μg/mL mouse IgG2b control (Cat# 400301; BioLegend) was added to the co-culture.
For the T cell proliferation assay, autologous CD8+ T cells were labeled with 0.5 μM CFSE (Cat# C34554; Thermo Fisher Scientific) for 15 min at room temperature and incubated with mature dendritic cells (DCs; 5:1) and the indicated labeled macrophages (2:1) in RPMI-1640 medium supplemented with 5 μg/mL IL-12, 25 mM HEPES, 4 mM L-glutamine, 25 μM 2-mercaptoethanol, and 10% FBS. Proliferation of CD8+ T cells was measured by CFSE staining and flow cytometry after 4 days. In some experiments, 5 μg/mL anti-human B7-H4 neutralizing Ab or 5 μg/mL mouse IgG2b control (Cat# 400301; BioLegend) was added to the co-culture.
3
Evaluating STAT3 Inhibition in CALR Cells
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Cell viability was measured with XTT assay according to the manufacturer’s protocol (Biological Industries, Beit Haemek, Israel). For the STAT3 inhibition assay, STAT3 Inhibitor S3I-201 (573102, Sigma-Aldrich, Macquarie, Australia) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 25 mM as the stock solution. CALRDEL cells were exposed to various concentrations of S3I-201 for 48 h, and the IC50 concentration was determined by XTT assay. Based on the obtained information, cells were treated with S3I-201 at a concentration of 25 µM that did not affect the cellular viability in subsequent STAT3 inhibition assays.
To assess cellular proliferation, cells cultured for 24 h were harvested for Ki67-staining (BioLegend, San Diego, CA, USA) and subsequent flow cytometric analysis. The percentages of apoptotic cells were determined using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) after the cells were treated with a 7-AAD and APC-Annexin V Apoptosis Detection Kit (BD Biosciences). All experiments were performed in triplicate, and only representative data were shown.
To assess cellular proliferation, cells cultured for 24 h were harvested for Ki67-staining (BioLegend, San Diego, CA, USA) and subsequent flow cytometric analysis. The percentages of apoptotic cells were determined using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) after the cells were treated with a 7-AAD and APC-Annexin V Apoptosis Detection Kit (BD Biosciences). All experiments were performed in triplicate, and only representative data were shown.
4
Modulating NK Cell Proliferation
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Autologous NK cells labeled with CTDR were cultured alone or co-cultured with macrophages with indicated treatments (2:1) in complete media (RPMI 1640 medium supplemented with 10% FBS) and stimulated with 100U/ml IL-2 and 50U/mL PeproTech) for 4 days. Afterward, the proliferation rate was evaluated by flow cytometry for Ki-67 staining (Cat# 350503, Biolegend) . In some experiments, 5 mg/ml anti-human PD-L1 neutralizing antibody (Cat# 329709, Biolegend), 5 mg/ml mouse IgG2b control (Cat# 400301, Biolegend) or 4 mg/ml 1-Methyl-D-tryptophan (1-MT, Cat# 452483, Sigma) was added in the co-culture.
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