Asynchronous cells were pulse-labelled for 15 min with EdU (5-ethynyl-2′-deoxyuridine) and then fixed and stained to visualize cells in the S phase as described by the manufacturer (Invitrogen). Cells were also labelled with MPM-2 monoclonal antibody (1:500, Abcam) to visualize mitotic cells. DNA was stained with 10 μg ml−1 propidium iodide in the presence of 0.1 mg ml−1 RNAse A (Sigma-Aldrich). Fluorescence was acquired using a MACS-Quant flow cytometer (Miltenyi), and data were processed in MACS-Quantify (Miltenyi) and Venturi-One (Applied Cytometry Systems) softwares. Cell ploidy was inferred from DNA contents in confluent quiescent cells. The growth curve was established from triplicate cultures initiated at the density of 2 × 103 cells per cm2 and counted every 24 h over 5 days using a MACS-Quant flow cytometer (Miltenyi).
Macsquant flow cytometer
Manufactured by Miltenyi Biotec
Sourced in Germany, United States, France
The MACSQuant flow cytometer is a compact and versatile instrument for cell analysis. It is designed to provide reliable and accurate measurements of various cellular parameters, including size, granularity, and fluorescence intensity. The MACSQuant flow cytometer utilizes state-of-the-art technology to deliver high-quality data for a wide range of applications.
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Lab products found in correlation
Macsquantify software, by Miltenyi Biotec (20 mentions)
Macsquant running buffer, by Miltenyi Biotec (7 mentions)
Trypsin edta, by Thermo Fisher Scientific (6 mentions)
Transwell chamber, by Corning (5 mentions)
Macsquantify analysis software, by Miltenyi Biotec (5 mentions)
Cxcl12, by R&D Systems (4 mentions)
Sytox green, by Thermo Fisher Scientific (4 mentions)
Anti cd24 pe, by BioLegend (3 mentions)
Anti cd44 apc, by BioLegend (3 mentions)
H2dcfda, by Merck Group (3 mentions)
Live dead fixable viability dye, by Thermo Fisher Scientific (3 mentions)
Fatty acid free bovine serum albumin faf bsa, by Merck Group (3 mentions)
Fixable yellow dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
Macsquantify, by Miltenyi Biotec (3 mentions)
Rbc lysis buffer, by BioLegend (3 mentions)
Celltrace violet, by Thermo Fisher Scientific (3 mentions)
Cxcl13, by R&D Systems (3 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Fxcycle pi rnase staining solution, by Thermo Fisher Scientific (3 mentions)
300 protocols using macsquant flow cytometer
1
Cell Cycle Dynamics Profiling
2
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, cells were harvested at the completion of the miRNAs treatments and washed with phosphate-buffered saline (PBS; pH 7.4) before being fixed with 70% ethanol on the wheel for 15 min at 4 °C. Subsequently, the cells were centrifuged at 4500 rpm for 5 min at 4 °C, washed with phosphate-buffered saline and were resuspended in 600 μl of 0,1% sodium citrate (Sigma-Aldrich), 50 μg/ml of propidium iodide (PI, Sigma-Aldrich) and 10 μg/ml of Ribonuclease A (Sigma-Aldrich) for staining cellular DNA. The cellular DNA content was then analyzed using a MACS Quant Flow Cytometer (Miltenyi Biotec). Data analysis was carried out using FlowJo software. For the apoptosis analysis, after treatment with miRNAs 10 nM, cells were washed with PBS and stained with Annexin V-FITC and PI using the Apoptosis Detection Kit (Bender Medsystems) according to the manufacturer’s protocol. Annexin-positive cells were counted using a MACS Quant Flow Cytometer (Miltenyi Biotec) within 1 h after staining. Data analysis was carried out using FlowJo software.
3
Viability and Immunophenotyping of hAMSCs
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To assess the viability, gene expression profile, and immunophenotype of hAMSCs adhered to the plastic around the CaP-coated samples, cells were preliminarily harvested with 0.05% trypsin (PanEco, Moscow, Russia) in 0.53 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) and washed twice with phosphate-buffered saline.
The surface markers on viable hAMSCs were analyzed with a Human MSC Phenotyping Kit (cat. no. 130-095-198, Miltenyi Biotec, Bergisch Gladbach, Germany), which detects the markers CD14, CD20, CD34, CD45, CD73, CD90, and CD105. After a 10-min incubation with the labeled monoclonal antibodies (mAbs), the cells were assayed using a MACS Quant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. The in vitro viability of hAMSCs was also estimated with the MACS Quant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) after staining with a solution of Annexin V: FITC (Abcam, Cambridge, UK) and propidium iodide (Abcam, Cambridge, UK) according to the manufacturer’s protocol. The flow cytometric data were analyzed using KALUZA analysis software (Beckman Coulter, Brea, CA, USA).
The surface markers on viable hAMSCs were analyzed with a Human MSC Phenotyping Kit (cat. no. 130-095-198, Miltenyi Biotec, Bergisch Gladbach, Germany), which detects the markers CD14, CD20, CD34, CD45, CD73, CD90, and CD105. After a 10-min incubation with the labeled monoclonal antibodies (mAbs), the cells were assayed using a MACS Quant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. The in vitro viability of hAMSCs was also estimated with the MACS Quant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) after staining with a solution of Annexin V: FITC (Abcam, Cambridge, UK) and propidium iodide (Abcam, Cambridge, UK) according to the manufacturer’s protocol. The flow cytometric data were analyzed using KALUZA analysis software (Beckman Coulter, Brea, CA, USA).
4
Macrophage Phagocytosis of Labeled RBCs
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After incubation at 37°C for 45 min, non-phagocytic RBCs were removed by washing three times with 2 ml of ice-cold isotonic saline (0Á9% NaCl). Residual RBCs were haemolysed with ice-cold hypotonic saline (0Á2% NaCl) for 2 min, followed by the addition of 2 ml of ice-cold hypertonic saline (1Á6% NaCl) for 2 min to restore isotonicity [13, 17] . Subsequently, the supernatant was removed and the adherent macrophage cells were detached by trypsin. Before and after trypsinization, THP cells were checked microscopically for remaining RBCs or detritus THP cells were diluted with 500 µl PBS (1 x 10 6 cells/ml) and kept on ice until analysis by flow cytometry (MACSQuant â Flow Cytometer, Miltenyi Biotech). At least 10 000 events were collected for each sample. Data were analysed using the FlowJo â software (FlowJo LLC, Ashland, OR, USA). The mean fluorescence and percentage of cells that were strongly positively stained for PKH26-redlabelled RBCs compared with control group were calculated. Controls using RBCs from two or three healthy blood donors with no selection of age, sex or blood group were included in each experiment.
5
Flow Cytometry of Activated THP-1 Cells
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After a 10-min trypsinization period (GibcoTM TrypLE Express), PMA-treated THP-1 cells were resuspended in cell culture medium. PMA-treated and non-treated THP-1 cells (0Á5 9 10 6 ) were diluted in 200 ll PBS and stained with 10 µl of the fluorescent labelled anti-human CD11b / MAC-1 (FITC) or anti-human HLA-DR (APC) antibodies (both from BD Biosciences Pharmingen TM). Cells were subsequently analysed by flow cytometry [16] with MACSQuant â Flow Cytometer (Miltenyi Biotech, Bergisch Gladbac, Germany).
6
Intracellular Cytokine Staining of Lymphocytes
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PB-, lymph node-, or spinal cord-infiltrating lymphocytes were prepared by Percoll gradient centrifugation and then incubated with Cell Stimulation Cocktail (eBioscience, San Diego, CA, USA) for 5 h. For intracellular staining, single-cell suspensions were prepared, surface-stained with FITC-labeled anti-CD4 antibody (Miltenyi Biotec, Germany), and then fixed and permeabilized using a Fixation/Permeabilization Kit (BD Biosciences, San Jose, CA, USA). Subsequently, the cells were washed and stained with Anti-IL-17-PE antibody (Miltenyi Biotec, Germany). Co-staining with RORγt, Foxp3, suppressor of cytokine signaling 3 (SOCS3) rabbit lgGs (CST, USA) was performed for some experiments, and APC-Goat anti-Rabbit lgG (Miltenyi Biotec, Germany) were used as secondary antibodies. Analyses were performed using a MACSQuantTM Flow Cytometer (Miltenyi Biotec, Germany), and the results were analyzed using FlowJo7.6. Protein expression levels are expressed as mean fluorescence intensity (MFI).
7
CD44 and CD24 Expression in Breast Cancer Cells
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MCF‐7 cells, T47D cells, MCF‐7 CSCs and T47D CSCs were digested with 0.25% trypsin, and they were then stained with anti‐CD44‐APC (Biolegend) (1.25 μL/test) and anti‐CD24‐PE (Biolegend) (5 μL/test) or negative controls at 4°C for 30 minutes. After staining, cells were washed three times with PBS and suspended in 300 μL of PBS. Flow cytometry analysis was performed on a MACSQuantTM Flow Cytometer (Miltenyi Biotec).
8
Quantifying Apoptosis in TPC-1 Cells
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We performed flow cytometry analysis to determine apoptotic rate of different experimental groups of TPC-1 cells using the Annexin V/Dead Cell Apoptosis Kit (Invitrogen) according to manufacturer’s instructions. The stained cells were analyzed using a MACSQuantTM Flow Cytometer (Miltenyi Biotec, USA) and the percentages of apoptotic (Annexin-V+) cells in each sample was determined using the Cell Quest software version 3.2 (BD Biosciences, USA).
9
Flow Cytometry Characterization of CD44/CD24 Expression
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All cell lines were digested with 0.25% trypsin and washed with PBS three times. Then cells were resuspended in 100 μL PBS and incubated with anti-CD44-APC (1.25 μl/ test) and anti-CD24-PE (5 μl/ test) (Bio legend, San Diego, USA), or with their controls at 4 °C for 30 min. After incubation, the cells were washed two times with PBS and suspended in 300 μL PBS. Analysis was performed on a MACSQuantTM Flow Cytometer (Miltenyi Biotec).
10
CD44 and CD24 Expression Analysis
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All cell lines were digested with 0.25% trypsin and washed with PBS three times. Then cells were resuspended in 100μL PBS and incubated with anti-CD44-APC (1.25 μl/ test) and anti-CD24-PE (5 μl/ test) (Biolegend, San Diego, USA), or with their controls at 4 °C for 30min. After incubation, the cell distribution was measured using a MACSQuantTM Flow Cytometer (Miltenyi Biotec).
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