To confirm the ploidy of the recipients, erythrocytes collected from the recipients were fixed in 70% (vol/vol) ethanol for 20 min at −20 °C and incubated for a period of 5 min at 25 °C in PBS (pH 7.8) that contained RNase A (10 μg ml−1; Sigma) and propidium iodide (200 μg mL−1; Sigma). After filtration through a 42-μm pore nylon screen, DNA content was analyzed using a Guava PCA-96 flow cytometry system (Millipore, Darmstadt, Germany).
Guava pca 96 flow cytometry system
Manufactured by Merck Group
Sourced in Germany
The Guava PCA-96 is a flow cytometry system designed for high-throughput analysis. It utilizes a compact and automated design to quickly process and analyze multiple samples. The system provides quantitative data on various cellular parameters, including size, granularity, and fluorescence intensity.
Automatically generated - may contain errors
4 protocols using guava pca 96 flow cytometry system
1
Ploidy Determination of Erythrocytes
2
Ploidy Analysis of Donor, Recipients, and Offspring
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To determine the ploidy level of the donor, recipients and F1 offspring, blood cells were fixed in 70% (vol/vol) ethanol and incubated for 8 h in PBS (pH 7.8) that contained RNase A (10 μg/ml; Sigma) and propidium iodide (200 μg/ml; Sigma). DNA contents were analyzed using a Guava PCA-96 flow cytometry system (Millipore). Mitotic chromosomes were made from fin and kidney cells of the donor and F1 offspring, respectively. Cells were treated with 0.4% (wt/vol) colchicine (Sigma) for 5 h, hypotonized in 0.075 M KCl (Gibco), fixed in methanol/glacial acetic acid (vol/vol; 3:1), air dried, and stained with 10% (vol/vol) Giemsa (Sigma) for 15 min as previously described40 (link). For each specimen, at least 30 countable metaphase chromosomes were examined under a light microscope (BX-53; Olympus).
3
Cell Viability Assay with Propidium Iodide
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Cells were plated at 3 × 10 5 cells/well in six-well plates and incubated for 24 h. Cultures were then treated with AA in Dulbecco's modified Eagle's medium for 4 h at 37°C and further incubated for 24 or 48 h in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum/pen-strep. After the recovery time, cells were labeled with propidium iodide (Sigma) using standard protocols and analyzed in a Guava® PCA-96 Flow Cytometry System (Millipore). The data were analyzed using the software Flowing Software 2.
4
Cryopreservation of Germ Cells
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Fresh and frozen ovaries were dissociated into single cells [24] , and the resultant cell suspensions were filtered through a 20-μm-pore nylon screen (Tokyo Screen Company, Japan). Because the total numbers of GFP (+) germ cells did not significantly differ between both ovaries of rainbow trout (n = 4, P < 0.05), the numbers of GFP (+) germ cells in frozen (1, 73, 273, 450, 642 , and 1,185 days) and fresh ovaries were compared to determine the viability of cryopreserved GFP (+) germ cells (n = 12-24).
Survival of GFP (+) germ cells was assessed using a Guava PCA-96 flow cytometry system (Millipore) and trypan blue (TB)-exclusion assay. The viability of GFP (+) germ cells was calculated as: viability (%) = ([number of GFP-positive and TB-negative cells in frozen ovary]/[number of GFP-positive cells in fresh ovary]) × 100.
Survival of GFP (+) germ cells was assessed using a Guava PCA-96 flow cytometry system (Millipore) and trypan blue (TB)-exclusion assay. The viability of GFP (+) germ cells was calculated as: viability (%) = ([number of GFP-positive and TB-negative cells in frozen ovary]/[number of GFP-positive cells in fresh ovary]) × 100.
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