For cytokine assay, splenocytes were distributed in a 24-well culture plate at a density of 1 × 106/ml and stimulated with 10 ug/ml VP1 protein for 72 h. These cells were also stimulated with 500 ng/ml Ionomycin (Sigma-Aldrich) and 50 ng/ml PMA (Sigma-Aldrich) plus 5 ng/ml Brefeldin A (eBioscience) at 37°C and 5% CO2 for the last 5 h. And then splenocytes were collected and stained with the following anti-mouse antibodies: PE-conjugated IFN-γ+, APC-conjugated CD8 (dilution 1:100, Biolegend, San Diego, CA). For detection of memory CD8 T cells, splenocytes from immunized mice were isolated and performed cell surface staining with the following anti-mouse antibodies: APC-conjugated CD8, FITC-conjugated CD44, and PE-conjugated CD62L (dilution 1:100, Biolegend, San Diego, CA). All flow cytometry data were acquired on a BD FACSCanto II (BD Biosciences) in FACSDiva software (BD Biosciences) and analyzed by FlowJo software (Tree Star Inc.).
Fitc conjugated cd44
Manufactured by BioLegend
FITC-conjugated CD44 is a flow cytometry antibody that binds to the CD44 protein, which is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. The FITC fluorescent label allows for the detection and analysis of CD44-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Pe conjugated cd62l, by BioLegend (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Apc conjugated ki 67 antibody, by BioLegend (1 mentions)
Percp cy5.5 conjugated cd4, by BioLegend (1 mentions)
Brilliant violet 510 conjugated cd8a, by BioLegend (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
α mem, by Cyagen (1 mentions)
Fitc conjugated cd45, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
3 protocols using fitc conjugated cd44
1
Cytokine Assay for Antigen-Specific CD8+ T Cells
2
Antigen-specific T cell proliferation assay
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Freshly isolated splenocytes (1 × 10E6) were seeded in 96-well plates and stimulated with 5 μg/mL Ag85B antigen or 1 μ/mL ESAT-6 antigen. As positive control, the cells were stimulated with 1 μg/mL α-CD3 (BioLegends). Antigen-specific T cell proliferation was analyzed after 6 days of incubation with antigens. The cells were blocked, as described above, and subsequently stained with PerCP/Cy5.5-conjugated CD4, Brilliant Violet 510™-conjugated CD8a, FITC-conjugated CD44, PE-conjugated CD62L and Brilliant Violet 421™- conjugated CD90.2 antibodies (all from BioLegends), all diluted 1:100. After staining, the cells were fixed using eBioscience™ Foxp3/Transcription Factor Staining Buffer Set and permeabilized using eBioscience™ Permeabilization Buffer, according to the manufacturer's instructions. Subsequently, cells were intracellularly stained with 1:50 diluted APC-conjugated Ki-67 antibody (BioLegends) and analyzed by flow cytometry.
3
Isolation and Osteogenic Differentiation of BMSCs
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After the second passage, the cells were collected and identified using fluorescently-labeled antibodies for BMSC markers: Allophycocyanin-conjugated CD29 (cat. no. 102225; BioLegend, Inc.); FITC-conjugated CD44 (cat. no. 203906; BioLegend, Inc.); FITC-conjugated CD45 (cat. no. 202205; BioLegend, Inc.); and phycoerythrin-conjugated CD34 (ab223930; Abcam). Briefly, the cells were incubated with corresponding antibodies for 30 min at 4°C in the dark and then washed with PBS. The expression levels of different cell surface markers were detected using FACSCalibur flow cytometer and analyzed using CellQuestPro version 5.1.1 software (BD Biosciences).
After detecting the purity, BMSCs from the second passage were used in subsequent experiments. To induce osteogenic differentiation, BMSCs were initially cultured in complete culture media as aforementioned. When the cells reached 80% confluence, osteogenic-inducing medium [α-MEM (Cyagen Biosciences) supplemented with 10% FBS, 50 mg/ml ascorbate, 10 mM β-glycerophosphate, 100 nM dexamethasone and 1% penicillin-streptomycin] was used. Lastly, the plates were stained with alizarin red and alkaline phosphatase at four time-points (0, 3, 7, and 14 days).
After detecting the purity, BMSCs from the second passage were used in subsequent experiments. To induce osteogenic differentiation, BMSCs were initially cultured in complete culture media as aforementioned. When the cells reached 80% confluence, osteogenic-inducing medium [α-MEM (Cyagen Biosciences) supplemented with 10% FBS, 50 mg/ml ascorbate, 10 mM β-glycerophosphate, 100 nM dexamethasone and 1% penicillin-streptomycin] was used. Lastly, the plates were stained with alizarin red and alkaline phosphatase at four time-points (0, 3, 7, and 14 days).
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