To analyze their DNA content, gametocytes were harvested in SA and purified as described above. Gametocytes were resuspended in 100 μl and activated with 100 μl of modified exflagellation medium [RPMI 1640 containing 25 mM Hepes, 4 mM sodium bicarbonate, 5% FCS, and 200 μM XA (pH 8.0)]. Gametogenesis was stopped by ice-cold PBS containing Vybrant dye cycle violet (Life Technologies), and cells were stained for 30 min at 4°C. DNA content was determined using a Beckman Coulter Gallios 4 and analyzed with the Kaluza software. Per sample, >10,000 cells were analyzed.
Gallios 4
Manufactured by Beckman Coulter
The Gallios 4 is a flow cytometer designed for automated analysis of cell samples. It features multiple laser configurations and a range of detection channels for comprehensive sample characterization. The Gallios 4 is capable of analyzing a variety of cell types and provides high-resolution data for research and clinical applications.
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5 protocols using gallios 4
1
Gametocyte DNA Content Analysis
2
Flow Cytometric Analysis of Virus-Induced Apoptosis
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Cells were seeded in 12-well plates at subconfluent density (70%) and infected with each virus at an MOI of 5 without TPCK-treated trypsin. Twenty-four hours postinfection, cells were harvested using trypsin-EDTA and processed through staining for apoptosis markers. For this protocol, all spins were performed for 5 min at 4°C at 400 rcf and all solutions were used at 4°C unless mentioned otherwise. Cells were washed twice with 1 ml of PBS, resuspended in 100 μl of PBS with 1 μl of Zombie Red fixable viability dye (BioLegend), and incubated 30 min at 4°C in the dark. Then cells were washed twice with 1 ml of PBS–2% BSA and once with annexin V binding buffer (BioLegend) at room temperature (RT). Cells were incubated for 15 min at RT with 5 μl of annexin V (BioLegend) diluted in 100 μl of annexin V binding buffer. Then cells were washed once in annexin V buffer and once in PBS supplemented with calcium and magnesium (PBS-Ca-Mg). Cells were fixed in 200 μl of 2% formaldehyde diluted in PBS-Ca-Mg for 20 min at RT. Cells were washed twice in PBS-Ca-Mg and then resuspended in 200 μl of PBS-Ca-Mg. Samples were analyzed by flow cytometry using a Gallios 4 (Beckman Coulter) and results were analyzed with the software Kaluza Analysis 1.5a.
3
Gametocyte DNA Content Analysis
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DNA content of microgametocytes was determined by flow cytometry measurement of fluorescence intensity of cells stained with Vybrant dye cycle violet (Life Technologies) as previously described (56 (link)). Gametocytes were purified and resuspended in 100 μl of SA. Activation was induced by adding 100 μl of modified exflagellation medium [RPMI 1640 containing 25 mM Hepes, 4 mM sodium bicarbonate, 5% FCS, and 200 μM XA (final pH 7.8)]. To rapidly block gametogony, 800 μl of ice-cold PBS was added and cells were stained for 30 min at 4°C with Vybrant dye cycle violet and analyzed using a Beckman Coulter Gallios 4. Gating was performed on both micro- and macrogametocytes, and cell ploidy was expressed as a percentage of all gametocytes. For each sample, >5000 cells were analyzed.
4
Determining Gametocyte DNA Content by Flow Cytometry
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DNA content of microgametocytes was determined by FACS measurement of fluorescence intensity of cells stained with Vybrant dye cycle violet (life Technologies). Gametocytes were purified and resuspended in 100 μl of SA. Activation was induced by adding 100 μl of modified exflagellation medium (RPMI 1640 containing 25 mM HEPES, 4 mM sodium bicarbonate, 5% FCS, 200 µM xanthurenic acid, pH 7.8). To rapidly block gametogenesis, 800 μl of ice cold PBS was added and cells were stained for 30 min at 4°C with Vybrant dye cycle violet and analysed using a Beckman Coulter Gallios 4. Microgametocytes were selected on fluorescence by gating on GFP positive microgametocytes when the 820cl1 or its derivatives were used. In this case ploidy was expressed as a percentage of male gametocytes only. When 2.34 or its derivatives were analysed, gating was performed on both micro- and macrogametocytes and ploidy was expressed as a percentage of all gametocytes. Per sample, >50.000 cells were analysed.
5
Gametocyte DNA Content Analysis
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DNA content of microgametocytes was determined by flow cytometry measurement of fluorescence intensity of cells stained with Vybrant dye cycle violet (life Technologies). Gametocytes were purified and resuspended in 100 μl of SA. Activation was induced by adding 100 μl of modified exflagellation medium (RPMI 1640 containing 25 mM HEPES, 4 mM sodium bicarbonate, 5% FCS, 200 µM xanthurenic acid, pH 7.8). To rapidly block gametogony, 800 μl of ice cold PBS was added and cells were stained for 30 min at 4°C with Vybrant dye cycle violet and analysed using a Beckman Coulter Gallios 4. Microgametocytes were selected on fluorescence by gating on GFP positive microgametocytes when the 820cl1 parasite line or its derivatives were used. In this case, cell ploidy was expressed as a percentage of male gametocytes only. When the 2.34 line or its derivatives were analysed, gating was performed on both micro- and macro-gametocytes and cell ploidy was expressed as a percentage of all gametocytes. For each sample, >20,000 cells were analysed.
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