HSPCs were harvested from expansion cultures at day 7, resuspended at 1 × 106 cells/mL in RoboSepTM Buffer and incubated with 2 µM MitoProbeTM JC-1 cationic dye (Thermo Fisher Scientific; dissolved in DMSO) at 37 °C/5% CO2 for 30 min as per manufacturer’s guidelines. For control sample, 50 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Thermo Fisher Scientific) was added simultaneously. Cells were washed, resuspended in RoboSepTM Buffer, and stored on ice until analysis. Samples were analysed on a Guava® easyCyte Single Sample flow cytometer, using 488 nm excitation with 530/590 nm emission filters. JC-1 is a lipophilic, cationic dye that can selectively enter mitochondria and reversibly change colour from green to red as the membrane potential increases. In healthy cells with high mitochondrial (ΔψM), JC-1 instinctively generates complexes known as J-aggregates (oligomers) with red fluorescence. Whereas, in unhealthy or apoptotic cells with low (ΔψM), JC-1 maintains its monomeric form with a green fluorescence.
Guava easycyte single sample flow cytometer
Manufactured by Merck Group
Sourced in United Kingdom
The Guava easyCyte Single Sample Flow Cytometer is a compact and reliable instrument designed for cell analysis. It utilizes flow cytometry technology to provide rapid and accurate data on various cellular parameters, including size, granularity, and fluorescence. The core function of this product is to enable researchers and scientists to perform efficient and versatile cell analysis in their laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Incytetm software, by Merck Group (2 mentions)
Incyte software, by Merck Group (2 mentions)
Carbonyl cyanide 3 chlorophenylhydrazone cccp, by Thermo Fisher Scientific (1 mentions)
Karyomax colcemid solution, by Thermo Fisher Scientific (1 mentions)
Muse cell cycle kit, by Merck Group (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Dioc6, by Merck Group (1 mentions)
Mouse fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Mitosoxtm red, by Thermo Fisher Scientific (1 mentions)
Sca 1 e13 161.7, by BioLegend (1 mentions)
B220 ra3 6b2, by Abcam (1 mentions)
10 protocols using guava easycyte single sample flow cytometer
1
Mitochondrial Membrane Potential Assay
2
Rhenium Compound PC-1 and miRNA146a Effects
3
Cell Cycle Analysis Utilizing Muse™ Kit
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For the cell cycle analysis, Muse™ Cell Cycle Kit (Merck, Kenilworth, NJ, USA) was used. Flp‐In™ T‐REx™ 293 cell lines expressing the corresponding transgene were grown on four 10‐cm plates per cell line until 50% confluency, at which point KaryoMAX Colcemid solution (Gibco, Dublin, Ireland) was added 1:100 to two plates per cell line for 6 h. Cells were harvested by trypsinization and fixed by resuspending in ice‐cold 70% EtOH. The final EtOH suspension was calculated to contain 2 million cells/ml. The resuspensions were kept at −20°C overnight. For cell counting, 300 μl of cell suspension was centrifuged at 450 g for 5 min and washed once with 1× PBS. Three hundred microliters of cell cycle reagent was added, and the solution was incubated at room temperature for 30 min in the dark. A total of 10,000 events were measured for each sample on Guava easyCyte single sample flow cytometer (Merck). The G2‐phase peak was defined using the KaryoMAX‐treated samples, while the results were obtained from untreated samples.
4
Clover Expression in Hap1 Cells
5
MitoTracker Staining of HSPCs
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HSPCS were harvested from expansion culture at day 7, resuspended in Advanced RPMI 1640 (1 × 106 cells/mL) and stained with 100 nM MitoTracker® Deep Red FM (Thermo Fisher Scientific) dissolved in DMSO at 37 °C/5% CO2 for 30 min as per the manufacturer’s guidelines. Cells were then washed in prewarmed dPBS, resuspended in Advanced RPMI and stored on ice until being examined using a Guava® easyCyte Single Sample Flow cytometer and analysed using InCyteTM software (Merck Millipore, Watford, UK).
6
Neutrophil Identification by DiOC6 Staining
7
Cell-Type Specific Immunophenotyping
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Labeling of cell-type specific surface antigens of cells isolated from the peritoneal cavity was performed as described recently (54 (link)). Briefly, after saturating unspecific binding sites with mouse FcR-blocking reagent (Miltenyi Biotech) (10% blocking solution in 0.5% BSA, 2 mM EDTA in PBS [magnetic-activated cell sorting [MACS] buffer]) for 10 min at 4 °C, cells were harvested by centrifugation (1,500 rpm, 5 min, 4 °C) and subsequently resuspended in MACS buffer, also used to dilute immune-phenotyping antibodies (T cells: CD3; B cells: B220; macrophages: CD11b, mast cells: CD117; neutrophils: Ly6-G), (working dilution 1:100, each). After an incubation of 20 min at RT, excess antibodies were eliminated by three washing steps with MACS buffer (1,500 rpm, 5 min, 4 °C). Flow cytometric analysis was performed with a Guava easy Cyte single sample flow cytometer (Merck-Millipore); isotype controls were used as references. Gates were adjusted on live cells and cell size. Resulting populations were analyzed for fluorescence signals. Data were analyzed using the InCyte software (Merck-Millipore). Sorting of CD117+ cells was performed with fluorescence-activated cell sorter Aria.
8
Assaying Mitochondrial ROS in HSPCs
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HSPCs were harvested from expansion cultures at day 7. Cells were resuspended at 1x106 cells/mL in RoboSepTM Buffer and incubated with 5 µM MitoSoxTM Red (Thermo Fisher Scientific; dissolved in DMSO) at 37 °C/5% CO2 for 30 min as per manufacturer’s instructions. After incubations, cells were washed, resuspended in 0.5 mL RoboSepTM Buffer and stored on ice until being examined using a Guava® easyCyte Single Sample flow cytometer and analysed using InCyteTM software (Merck Millipore, Watford, UK).
9
Multicolor Flow Cytometry of Spleen Cells
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Spleen cells were incubated with phycoerythrin (PE)-conjugated antibodies: Sca1 (E13-161.7; BioLegend), cKit (2B8; Abcam, Cambridge, UK), Flt3 (A2F10.1), Gr1 (1A8), Ly6c (HK1.4; Abcam), Mac1 (M1/70), CD31 (MEC13.3), CD3 (17A2), and B220 (RA3-6B2). All reagents were purchased from BD Biosciences, unless otherwise stated. Acquisition was performed using a Guava® easyCyte Single Sample flow cytometer, and analyzed using InCyte™ software (Merck Millipore, Watford, UK).
10
Clover Expression in Hap1 Cells
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pcDNA3.1 CMV-ATG-Clover constructs were transfected into Hap1 wildtype and RPS25 knockout cells with Lipofectamine 3000. After 48 hours of transient transfection, Hap1 cells were dissociated and resuspended in 1X PBS, 2% FBS, 1mM EDTA buffer and analyzed in the FITC channel for GFP expression using a Guava easyCyte Single Sample Flow Cytometer (EMD Millipore). Data was analyzed using Flowjo (version X 10.0.7r2) and the mean GFP signal was calculated.
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