For the analysis of phagocytosis, spleen leukocytes were seeded in 24-well plates (Nunc) at a cell density of 1 × 106 cells per well and incubated for 24 h at 20°C with BAFF (3 μg/ml), TNP-LPS (5 μg/ml), a combination of both, or left unstimulated (control), and then incubated for another 16 h at 20°C with fluorescent polystyrene-based particles (FluoSpheres® Microspheres, 1.0 μm, Crimson Red Fluorescent 625/645, 2% solids; Life Technologies) at a cell:bead ratio of 1:10 or without beads as negative controls. Cells were harvested using a standard cell scraper (Corning). Non-ingested beads were removed by centrifugation (100 × g for 10 min at 4°C) over a cushion of 3% (weight/volume) bovine serum albumin [(BSA), Fisher Scientific] in PBS supplemented with 4.5% (weight/volume) d -glucose (Sigma). Cells were resuspended in staining buffer, labeled with an anti-IgM mAb fluorescently labeled with FITC, and analyzed on a FACSCalibur flow cytometer equipped with CellQuest sofware (BD Biosciences). The analysis was also performed with FlowJo 10.
Crimson red fluorescent 625 645
Manufactured by Thermo Fisher Scientific
The Crimson Red Fluorescent 625/645 is a fluorescent dye that is designed to emit light in the red portion of the visible spectrum. It has an excitation maximum of 625nm and an emission maximum of 645nm. This dye can be used in various biological and analytical applications that require the detection of fluorescent signals in the red wavelength range.
Automatically generated - may contain errors
Lab products found in correlation
D glucose, by Merck Group (5 mentions)
Fluospheres microspheres, by Thermo Fisher Scientific (4 mentions)
24 well plate, by Thermo Fisher Scientific (3 mentions)
Fraction 5, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Standard cell scraper, by Corning (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Bsa fraction 5, by Thermo Fisher Scientific (1 mentions)
5 protocols using crimson red fluorescent 625 645
1
Analysis of Phagocytosis in Spleen Leukocytes
2
Splenocyte Endocytosis Assay
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Purified splenocytes (2 × 106) were cultured in 1 ml/well in 24-well plates in the presence or absence of IL-6 or LPS for 24, 48 or 72 h. After each time point, cells were incubated for 16 h at 20 °C with fluorescent beads (FluoSpheres Microspheres, 1.0 μm, Crimson Red Fluorescent 625/645, 2% solids; Life Technologies) at a cell/bead ratio of 1:10 or without beads as negative controls. Non-ingested beads were removed by centrifugation (100 × g for 10 min at 4 °C) over a cushion of 3% (w/v) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5 (w/v) D-glucose (Sigma). Afterwards, cells were resuspended in staining buffer, labeled with PE-anti-IgM mAb (30 min at 4 °C), washed with the same buffer and analyzed by flow cytometry.
3
Quantifying Intestinal CD8+ DC Phagocytosis
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To analyze the phagocytic capacity of intestinal CD8+ DCs, leukocytes from the intestine were seeded in 24-well plates (Nunc) at a cell density of 1 × 106 cells per well and incubated for 16 h at 20 °C with fluorescent beads (FluoSpheres® Microspheres, 1.0 μm, Crimson Red Fluorescent 625/645, 2% solids; Life Technologies) at a cell:bead ratio of 1:10 or without beads in the case of negative controls. After the incubation period, cells were harvested by gently pipetting and non-ingested beads were removed by centrifugation (100×g for 10 min at 4 °C) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) d -glucose (Sigma). Cells were resuspended in L-15 with 5% FCS, labelled with the flow cytometry antibodies as described above and analyzed on a FACSCalibur flow cytometer. Flow cytometry analysis was performed using Flow Jo 10 software package.
4
Assessing Gill CD8+ DC Phagocytosis
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To analyze the phagocytic capacity of gill CD8+ DCs, gill leukocytes were seeded in 24-well plates (Nunc) at a cell density of 1 × 106 cells/well and incubated for 16 h at 20°C with fluorescent beads (FluoSpheres® Microspheres, 1.0 µm, Crimson Red Fluorescent 625/645, 2% solids; Life Technologies) at a cell:bead ratio of 1:10 or without beads in the case of negative controls. After the incubation period, cells were harvested by gently pipetting, and non-ingested beads were removed by centrifugation (100 × g for 10 min at 4°C) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) d -glucose (Sigma). Cells were resuspended in L-15 with 5% FCS, labeled with the flow cytometry antibodies and analyzed on a FACSCalibur flow cytometer or under the confocal microscope. In some experiments, cytochalasin B (0.05 µg/ml) was added to the cells immediately before the addition of the beads to verify active phagocytosis.
5
Phagocytosis Assay with Vitamin C
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For the analysis of phagocytosis, RTS11 cells or head kidney leukocytes were seeded in 96-well plates at a cell density of 2x10 5 cells per well and incubated for 3 h at 20ºC with fluorescent beads (FluoSpheres® Microspheres, 1.0 µm, Crimson Red Fluorescent 625/645, 2% solids; Life Technologies) at a cell:bead ratio of 1:10 in the presence or absence of different vitamin C doses (0.1, 0.5 and 1 M). Cells were harvested and non-ingested beads were removed by centrifugation (100 x g for 10 min at 4 ºC) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) D-glucose (Sigma). Cells were resuspended from the pellet in staining buffer (PBS containing 1% FCS and 0.5% sodium azide), labelled with anti-IgM [1.14 mAb mouse IgG 1 coupled to fluorescein (FITC), 1 μg/ml] as previously described [24] when needed, and analyzed on a FACSCalibur flow cytometer. In another set of experiments, the cells were previously incubated with the different vitamin C doses for 24 h at 20ºC before the addition of the fluorescent beads. Afterwards, the experiments were conducted as described above.
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