MV4-11 was primed with amiloride (10 μM) for 30 min and subjected to doxorubicin and ibrutinib uptake assay. For doxorubicin, amiloride-treated MV4-11 was cultured with doxorubicin (1 μM) for 1 hour and washed with PBS twice. The fluorescent intensity of intracellular doxorubicin was measured by CytoFLEX Flow Cytometer (Ex: 561 nm, Em: 585 nm). For ibrutinib, amiloride-treated MV4-11 was cultured with ibrutinib (1 μM) for 1 hour. The cell was then washed with PBS twice and cultured with PCI-33380 (10 μM), a fluorescent probe of BTK inhibitor. Cells were then washed with PBS twice and the fluorescent intensity of intracellular PCI-33380 was measured by CytoFLEX Flow Cytometer (Ex: 488 nm, Em: 525 nm).
Cytoflex flow cytometer
Manufactured by Beckman Coulter
Sourced in United States, Germany, Italy, China, Canada, Australia, Switzerland, United Kingdom, Japan, France, Belgium
The CytoFLEX flow cytometer is a versatile instrument designed for high-performance cell analysis. It utilizes advanced optical and fluidic technologies to enable accurate and reliable detection and enumeration of a wide range of cell populations. The CytoFLEX is capable of simultaneous multi-parameter analysis, providing researchers with comprehensive data on cellular characteristics and populations.
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Lab products found in correlation
Cytexpert software, by Beckman Coulter (141 mentions)
Cytexpert 2, by Beckman Coulter (44 mentions)
Propidium iodide, by Merck Group (32 mentions)
Cytexpert, by Beckman Coulter (23 mentions)
Kaluza software, by Beckman Coulter (18 mentions)
Golgistop, by BD (17 mentions)
Cytexpert v2, by Beckman Coulter (17 mentions)
Propidium iodide, by Thermo Fisher Scientific (14 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (13 mentions)
Bovine serum albumin (bsa), by Merck Group (12 mentions)
Cytofix cytoperm, by BD (12 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (12 mentions)
Triton x 100, by Merck Group (10 mentions)
Cytoflex, by Beckman Coulter (10 mentions)
True nuclear transcription factor buffer set, by BioLegend (10 mentions)
Aldefluor kit, by STEMCELL (9 mentions)
Kaluza analysis software, by Beckman Coulter (9 mentions)
Golgistop kit, by BD (9 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (9 mentions)
Cytofix cytoperm kit, by BD (9 mentions)
1 941 protocols using cytoflex flow cytometer
1
Amiloride Modulates Doxorubicin and Ibrutinib Uptake
2
Analyzing Tumor Immune Profiles
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The primary tumors were covered with 2‐cm chicken breast tissues and then treated with US for 10 min. After 10 days of treatments, the mice were euthanized to extract primary and distant tumors and tumor‐draining lymph nodes. These tissues were used to prepare single cell suspensions by grinding and filtering. The collected single cells were stained with antibodies, and then analyzed using a CytoFLEX flow cytometer. The tumor tissues were homogenized in PBS solution and the formed suspensions were filtered via cell strainers to obtain the single cell suspensions. The cells were stained with antibodies and then analyzed using a CytoFLEX flow cytometer.
3
Antifungal Activity Assays in C. albicans
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C. albicans cells pretreated with histatin 5, melittin, or HKK peptides at MICs for 6 h were incubated with 10 μM DCFH-DA for 20 min in the dark. DCF fluorescence was measured using 5000 events/sample following excitation with a 488-nm wavelength laser and reading through a 525/40 biopass filter using a CytoFLEX flow cytometer.
Mitochondrial SOX was measured in C. alibicans cells using a MitoSOX Red probe. Fungal cells were pre-incubated with histatin 5, melittin, or HKK peptides at MICs for 6 h, and fluorescent staining was performed following the manufacturer’s protocol for MitoSOX Red. The cells emitting red fluorescence were counted using a CytoFLEX flow cytometer.
Mitochondrial SOX was measured in C. alibicans cells using a MitoSOX Red probe. Fungal cells were pre-incubated with histatin 5, melittin, or HKK peptides at MICs for 6 h, and fluorescent staining was performed following the manufacturer’s protocol for MitoSOX Red. The cells emitting red fluorescence were counted using a CytoFLEX flow cytometer.
4
Flow Cytometric Analysis of B Cell Markers
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Flow antibodies (Biolegend) were used at 1/50 dilution to stain cell samples in PBS (Supplementary Table 2 ). Samples were measured using the Cytoflex flow cytometer (Beckman Coulter). Surface antigen expression (BAFF-R, BCMA, TACI, CD19) was characterized using the Cytoflex flow cytometer (Beckman Coulter) and respective Flow antibodies (Biolegend).
5
Isolation and Analysis of Tumor-Infiltrating and Splenic T Cells
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For harvesting cells from the CT-26 and 4T1 tumor, the tumor was dissected out, cut into small pieces. The tumor tissues were dissociated using the Miltenyi mouse tumor dissociation kit according to the manufacturers’ instructions. The preparations were passed through a 70 μm cell strainer and washed thoroughly with PBS buffer supplemented with 0.5% BSA (PBS-BSA buffer). Finally, the cells were resuspended in PBS-BSA buffer and stained with CD8 alpha Monoclonal Antibody (KT15). FITC (for CD8+ T cells) or CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), Super Bright 780, eBioscience™ (for PD-L1 expression levels) for flow cytometry on Beckman Coulter CytoFLEX flow cytometer and analyzed using FlowJo. For harvesting cells from spleen tissues, the spleen was minced finely with scissors and scalpel, and mashed on a 70 μm cell strainer to create a single-cell suspension. The red blood cells were lysed by incubating cells in ACK lysis buffer for 10 minutes and mononuclear cells were washed thoroughly with PBS-BSA buffer. Finally, the cells were resuspended in PBS-BSA buffer and stained with CD8 alpha Monoclonal Antibody (KT15). FITC (for CD8+ T cells) for flow cytometry on Beckman Coulter CytoFLEX flow cytometer and analyzed using FlowJo.
6
Isolation and Analysis of Tumor-Infiltrating and Splenic T Cells
7
Flow Cytometry Analysis of ACE2 and Apoptosis
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For flow cytometry analysis of cell surface ACE2, 100 μL of HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells (1 × 106) was digested to a single-cell suspension, and incubated with primary rabbit anti-human ACE2 antibodies followed by FITC-conjugated IgG secondary antibodies. Rabbit IgG was used as the isotype control. Cells were then washed thrice with centrifugation at 400g and analysed using a FC500 flow cytometer (Beckman, Germany) or CytoFLEX flow cytometer (Beckman). For apoptosis analysis, HEK293T, HEK293T-hACE2, Vero E6, 16HBE, and HMEC-1 cells (1 × 105) treated with or without SARS-CoV-2 spike pseudovirions or recombinant spike proteins (10 μg/mL) in parallel with the addition of an equal volume of DMSO or 3-MA (10 mM) for 24 h were labeled with FITC-conjugated Annexin V and 7-AAD (Biolegend, USA) in the dark for 15 min and analysed using an FC500 flow cytometer (Beckman) or CytoFLEX flow cytometer (Beckman).
8
Cell Apoptosis and Cell Cycle Analysis of 4T1 Cells
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For cell apoptosis assay, 4T1 cells in logarithmic growth phase were seeded at a density of 5 × 105 cells per well in a 6-well plate and incubated overnight at 37 °C. The cells were treated with MSNs with different shapes at an equivalent PTX concentration of 20 μg/mL for 48 h in triplicate with untreated cells serving as controls. After treatment, the cells were harvested by centrifugation and washed twice with cold PBS. Subsequently, the cells were resuspended in an equal volume of binding buffer, followed by staining with 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) solution at room temperature for 15 min. The cell cycle was detected using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA).
For cell cycle analysis, 4T1 cells in the logarithmic growth phase were seeded at a density of 5 × 105 cells per well in a 6-well plate and incubated overnight at 37 °C. The cells were treated with different shapes of MSN and PTX solution for 24 h in triplicate with untreated cells serving as a control. The cells were then precipitated, washed with ice-cold PBS, and fixed overnight at 4 °C in 70% (v/v) ethanol. Subsequently, the cells were centrifuged, washed with PBS, and stained with 500 μL propidium iodide (PI) solution at 37 °C for 30 min. The cell cycle was detected using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA) [42 (link)].
For cell cycle analysis, 4T1 cells in the logarithmic growth phase were seeded at a density of 5 × 105 cells per well in a 6-well plate and incubated overnight at 37 °C. The cells were treated with different shapes of MSN and PTX solution for 24 h in triplicate with untreated cells serving as a control. The cells were then precipitated, washed with ice-cold PBS, and fixed overnight at 4 °C in 70% (v/v) ethanol. Subsequently, the cells were centrifuged, washed with PBS, and stained with 500 μL propidium iodide (PI) solution at 37 °C for 30 min. The cell cycle was detected using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA) [42 (link)].
9
Quantifying Viral and Bacterial Abundance
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Viral abundance was estimated following the previous methods (62 (link), 63 (link)) with some modifications. The fixed and frozen samples (−80°C) were thawed at 37°C, diluted 10- to 100-fold with 0.02-μm filtered Tris-EDTA buffer (pH 8) (Sigma-Aldrich, Hamburg, Germany) and stained with SYBR green I (final concentration of 0.5 × 10−4 of the Molecular Probes stock solution, Thermo Fisher, USA) in the dark at 80°C. Then, the incubated samples were cooled at room temperature for 5 min and analyzed with a CytoFLEX flow cytometer (Beckman, Shanghai, China) at a total volume of 30 μL sample−1. For bacterial enumeration (63 (link), 64 (link)), the thawed samples were diluted 10-fold with 0.02-μm filtered Tris-EDTA buffer, stained with SYBR Gold (final dilution of 10−4 of the commercial stock solution) for 15 min in the dark, and analyzed with the CytoFLEX flow cytometer (Beckman) for 1 min at a delivery rate of 60 μL min−1.
10
Apoptosis and Cell Cycle Analysis
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Apoptosis was detected using an Annexin V-FITC/PI double-staining apoptosis detection kit (KeyGen Biotech, KGA107) according to manufacturer instructions. Cells were collected after treatment with trypsin without EDTA (Solarbio, T1350) and washed once with PBS. The cell pellet was resuspended in binding buffer, and 5 μL Annexin V-FITC and PI staining agents were added and incubated for 15 and 5 min in the dark, respectively. Then, the apoptosis rate of each group was determined with data obtained with a CytoFLEX Flow Cytometer (Beckman Coulter).
For cell-cycle analysis, cells were fixed in 70% ice-cold ethanol overnight. Next, 30 μL RNase A (Epicenter, RNR07250) was incubated with the cells at 37°C for 30 min. The cells were then stained at 4°C for 30 min with 120 μL PI (KeyGen Biotech, KGA512) in the dark. Fluorescence intensity was measured using a CytoFLEX Flow Cytometer (Beckman Coulter). The proportion of cells in the G0/G1, S, and G2/M cell-cycle phases was determined, and the proportions were then compared.
For cell-cycle analysis, cells were fixed in 70% ice-cold ethanol overnight. Next, 30 μL RNase A (Epicenter, RNR07250) was incubated with the cells at 37°C for 30 min. The cells were then stained at 4°C for 30 min with 120 μL PI (KeyGen Biotech, KGA512) in the dark. Fluorescence intensity was measured using a CytoFLEX Flow Cytometer (Beckman Coulter). The proportion of cells in the G0/G1, S, and G2/M cell-cycle phases was determined, and the proportions were then compared.
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