Synaptosomes were treated with AβO and/or TauO for binding challenges, and the binding percentages were evaluated with flow cytometry. After assessing that the binding of AβO and/or TauO was comparable among the single human cases (Supplementary method 2), we decided to pool together an equal number of synaptosomes isolated from each subject for practical purpose. We incubated 2 million of synaptosomes for 1 h at RT without oligomers (control) as well as with AβO tagged with HyLite Fluor 647 or/and TauO tagged with Alexa Fluor™ 488 NHS Ester (Thermo Fisher Scientific) at concentrations of 0–0.5–1–2.5–5–10 μM. Synaptosomes were then pelleted, washed three times with HBK buffer, and resuspended in HBK. Oligomer fluorescence positivity was acquired by a Guava EasyCyte 8 flow cytometer (EMD Millipore) and analyzed using Incyte software (EMD Millipore).
Guava easycyte 8 flow cytometer
Manufactured by Merck Group
Sourced in United States, Germany
The Guava easyCyte 8 is a flow cytometer that measures and analyzes the physical and chemical characteristics of cells or particles suspended in a fluid stream. It is designed to provide fast and efficient cell analysis for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Rnase a, by Takara Bio (2 mentions)
Propidium iodide, by Keygen Biotech (2 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (2 mentions)
Ghost dye red 780, by Cytek Biosciences (2 mentions)
Hcx 63, by Leica (2 mentions)
Tcs sp5 confocal microscope, by Leica (2 mentions)
6 well plate, by Sarstedt (2 mentions)
Modfit software 5, by Verity Software House (2 mentions)
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
Incyte software, by Merck Group (1 mentions)
Guava incyte software, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
2 nbdg, by Merck Group (1 mentions)
2 nbdg, by Apexbio (1 mentions)
Facscalibur, by BD (1 mentions)
Alexa fluor 647, by Beyotime (1 mentions)
Beyoclick edu cell proliferation kit, by Beyotime (1 mentions)
Fitc conjugated anti cd34 antibody, by Thermo Fisher Scientific (1 mentions)
Fc blocker, by BD (1 mentions)
29 protocols using guava easycyte 8 flow cytometer
1
Synaptosomes Oligomer Binding Assay
2
Cell Cycle Analysis of MCF-7 and MDA-MB-231 Cells Treated with PBN
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MCF-7 and MDA-MB-231 cells were seeded in 6-well plates at a density of 8 × 104 cells per well and incubated for 24 h prior to treatment with 0.5 mM or 1 mM PBN. Cells were harvested at 24, 48, and 72 h following treatment, washed twice with phosphate-buffered saline (PBS), centrifuged at 200× g for 5 m at 4 °C, re-suspended in 1 mL of old PBS and fixed in 4 mL of cold absolute ethanol. Prior to flow cytometry analysis, cells were pelleted and treated with 100 µL of 200 µg/mL DNase-free RNase A for an hour at room temperature, and then incubated with 20 μg/mL propidium iodide ([PI]; Sigma) for 15 m in the dark. Cell cycle analysis was performed using a Guava Easy Cyte 8™ flow cytometer operated by Guava InCyte™ software (EMD Millipore, Darmstadt, Germany).
3
Cell Cycle Analysis by Flow Cytometry
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Cells were separately harvested 0, 24, 48 and 72 h after siRNA transfection, fixed in 70% ethanol, and stained with propidium iodide (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China) containing RNase A (1 mg/ml; Takara Biotechnology Co., Ltd.) for 30 min at 37°C. Subsequently, 500 µl of cells was filtered through 200-µm mesh sieves, and the cell cycle profiles were assayed using a Guava easyCyte 8 Flow Cytometer (EMD Millipore, Billerica, MA, USA).
4
Evaluating Candida krusei Morphology
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Candida krusei cells (105 CFU/ml) were treated in RPMI 1,640 buffered with hydrochloric acid to pH 4 with either 10 µM 3,6-PIRAMICAR or 250 µM fluconazole under the agitation of 180 rpm for 4 h at 37 °C. After incubation, the cells were harvested by centrifugation (1,000 rpm, 5 min 4 °C) and resuspended in 0.05 mM potassium phosphate buffer (pH 7.0). Morphological changes of cells induced by treatment were assessment by illuminating cells by 480 nm light from an argon ionic laser and determining their position on a forward scatter (FSC) versus side scatter (SSC) contour plot using the Guava easyCyte 8 flow cytometer (Merck Millipore).
5
Apoptosis Analysis of Glioma Cells
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Glioma cells were transfected with the appropriate oligonucleotides and incubated for 72 h in an incubator under 5% CO2 at 37°C in six-well plates. The cells were harvested with pancreatic enzymes without EDTA and washed twice with precooled PBS. The cells were resuspended at a concentration of 1 × 105 cells/ml in 500 μl of binding buffer containing 5 μl of annexin V-fluorescein isothiocyanate (FITC) and 5 μl of propidium iodide (PI), according to the instructions of the manufacturer of the Annexin V-FITC Apoptosis Detection Kit (KeyGEN, Shanghai, China). After incubation for 15 min at room temperature in the dark, all the samples were analyzed within 1 h with a guava easyCyte 8 flow cytometer (EMD Millipore, USA). The data were analyzed with the De Novo software.
6
Cell Cycle Analysis of sh-lncRNA-XIST Cells
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sh-lncRNA-XIST cells and sh-NC cells were seeded in 35 mm-petri plates at a density of 1x105cells/mL. After 24 h of incubation, cells were harvested and fixed in 70% ethanol, then stained with propidium iodide (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China) containing RNase A (1 mg/ml; Takara Biotechnology Co., Ltd.) for 30 min at 37˚C. Subsequently, 500 μL of cells was filtered through 200‑μm mesh sieves and the cell cycle profiles were assayed using a Guava easyCyte 8 Flow Cytometer (EMD Millipore, Billerica, MA, USA).
7
Glucose Uptake Measurement in hESC-ECs
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Glucose uptake was measured using the fluorescence-labeled deoxyglucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG, Apex Bio, USA). After the indicated treatment, hESC-ECs were incubated with 2-NBDG for 1 h at 37 °C, and the fluorescence intensity was measured using the Guava EasyCyte™ 8 flow cytometer (EMD Millipore, Germany).
8
Lysotracker-based Quantification of Adipocyte Lysosomes
9
Cell Cycle and Apoptosis Analysis
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A total of 1×104 cells/plate were transiently transfected for 24 h. The cells were then collected and fixed in 70% ethanol overnight at −20°C. Following washing, the fixed cells were incubated in PBS with FxCycle™ PI/RNase Staining Solution (cat. no. F10797; Invitrogen; Thermo Fisher Scientific, Inc.) with RNase A (0.5 mg/ml) and propidium iodide (PI; 50 µg/ml) for 30 min at 37°C. The percentage of cells in each phase of the cell cycle or apoptosis was quantified using a Guava® easyCyte 8 flow cytometer (EMD Millipore) with ModFit software 5.0 (Verity Software House). Blue sub-G1 accumulation represented apoptotic cells containing only fractional DNA content. The cells in each group were quantified based on three independent experiments.
10
Cell Cycle Analysis and Stem Cell Marker Profiling
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Cells were seeded (1×104 cells/plate in complete medium) for 24 h. Subsequently, the cells were treated with 10 µM pimozide, 20 µM bromocriptine or combined treatment for 24 h. The collected cells were fixed in 70% ethanol overnight at −20°C. Then fixed cells were washed, and incubated in PBS with FxCycle™ PI/RNase Staining Solution (cat. no. F10797 Invitrogen; Thermo Fisher Scientific, Inc.) with RNase A (0.5 mg/ml) and propidium iodide (PI; 50 µg/ml) for 30 min at 37°C. The percentage of cells in each phase of the cell cycle was quantified using a Guava® easyCyte™ 8 flow cytometer (EMD Millipore) with ModFit software 5.0 (Verity Software House). For cell marker analysis, cells were incubated with anti-CD133, anti-nestin or anti-Sox2 antibodies conjugated to fluorochromes for 30 min at 37°C and analyzed using a flow cytometric system (FACSCalibur; BD Biosciences). Gates were set on the basis of staining with isotype controls (product code ab125938; Abcam) so that no more than 0.1% of cells were detected with control antibodies. The cells in each group were quantified based on three independent experiments.
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