Blood, spleen, and BM cells from hu-mice were stained with antibodies and analyzed using a BD Accuri C6 Plus (BD Biosciences). The antibodies used to detect human cells included APC-conjugated anti-human CD45 (555485, BD PharmingenTM), FITC-conjugated anti-mouse CD45 (553080, BD PharmingenTM), APC-conjugated anti-human CD4 (555349, BD PharmingenTM), PE-conjugated anti-human CD8 (555367, BD PharmingenTM), PE-conjugated anti-human CD19 (302208, BioLegend), and PE-conjugated anti-human CD33 (303403, BioLegend). For immunocy-tochemistry, anti-CD45 (ab8216, Abcam) and anti-CD34 (MAB72271, R&D Systems) antibodies were used, and proper isotype-matched IgG antibodies were used to detect the primary signals. Flow cytometric data were analyzed using the CSamplerTM Plus software program (BD Bio-sciences).
Fitc conjugated anti mouse cd45
Manufactured by BD
Sourced in United States
The FITC-conjugated anti-mouse CD45 is a laboratory reagent used to detect and analyze mouse CD45-positive cells. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of most hematopoietic cells. The FITC (Fluorescein Isothiocyanate) conjugation allows for the visualization and detection of CD45-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Ab8216, by Abcam (1 mentions)
Pe conjugated anti human cd8, by BD (1 mentions)
Pe conjugated anti human cd33, by BioLegend (1 mentions)
Apc conjugated anti human cd45, by BD (1 mentions)
Pe conjugated anti human cd19, by BioLegend (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Facs flow cytometer, by Beckman Coulter (1 mentions)
Apc conjugated anti mouse cd25, by BD (1 mentions)
Pe conjugated anti mouse foxp3, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti mouse cd11c, by BD (1 mentions)
Pe conjugated anti mouse il 17a, by BD (1 mentions)
Easyseptm mouse na ve cd4 t cell isolation kit, by STEMCELL (1 mentions)
Apc conjugated anti mouse cd4, by BD (1 mentions)
Pe cy7 conjugated anti mouse cd3, by BioLegend (1 mentions)
Rag1 mice, by Jackson ImmunoResearch (1 mentions)
Facsaria 2, by BD (1 mentions)
Cell strainer, by BD (1 mentions)
Guava easycyte ht instrument, by Merck Group (1 mentions)
Pe conjugated anti mouse cd49b, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
5 protocols using fitc conjugated anti mouse cd45
1
Immunophenotyping of Humanized Mice
2
Multiparametric Analysis of Immune Cell Subsets
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For Treg detection, cells were first stained with FITC-conjugated anti-mouse CD45 (BD, United States), PE-CY-7-conjugated anti-mouse CD4 (BD, United States), and APC-conjugated anti-mouse CD25 (BD, United States). For Th17 cell detection, cells were stimulated with Cell Stimulation Cocktail in culture medium for 4 h before surface staining using FITC-conjugated anti-mouse CD45 and PE-CY-7-conjugated anti-mouse CD4. After surface staining, cells were fixed and permeabilized with fixation/permeabilization buffer, followed by staining with PE-conjugated anti-mouse Foxp3 (eBIOSCIENCE, United States) for Tregs and PE-conjugated anti-mouse IL-17A (BD, United States) for Th17 cells. For dendritic cells detection, cells were stained with FITC-conjugated anti-mouse CD11c (BD, United States). Cells were analyzed on a FACS flow cytometer (CytoFLEX, Beckman, United States and BD Biosciences, United States).
3
Adoptive Transfer of Naïve CD4+ T Cells Induces Colitis in Rag1-/- Mice
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C57BL/6 and Rag1−/− mice were purchased from Jackson Laboratory. All mice were kept in sterilized, ventilated cages under SPF conditions and fed either a control diet or exposed to AR via the diet. Mice were allowed to acclimatize for 7 days prior to the start of experiments. Colitis was induced in Rag1−/− mice by adoptive transfer of FACS-sorted CD4+CD45RBhi T cells. Naïve CD4+ T cells were isolated from splenocytes of C57BL/6 mice by EasySepTM Mouse Naïve CD4+ T cell Isolation Kit (StemCell Technology, Vancouver, Canada). Naïve CD4+ T cells were labeled with PE-cy7-conjugated anti-mouse CD3 (BioLegend; 1:100), APC-conjugated anti-mouse CD4 (BD Biosciences; 1:100), and FITC-conjugated anti-mouse CD45 (BD Biosciences; 1:100). CD4+CD45RBhi T cells were sorted using FACS Aria II flow cytometer (BD Biosciences; FACSDiva v 6.1.2). Cell viability was assessed using Trypan blue assay prior to injection. Recipients were intraperitoneally (i.p.) injected with 5 × 105 cells of sorted T cells. Samples from all mice were kept at −80 °C.
4
Phenotypic Characterization of BMSCs
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Two groups of cells, BMSCs group and CXCR-BMSCs group, were trypsinized, inactivated with FBS and washed twice with PBS. These cells were suspended in 100 µl PBS and incubated at 4°C for 20 min with PE-conjugated anti-mouse CD90.2 (BD Biosciences, Franklin Lakes, NJ, USA), Alexa Fluour-conjugated anti-mouse CD105 (BD Biosciences), PE Cy™-conjugated anti-mouse CD11b (eBioScience; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and FITC-conjugated anti-mouse CD45 (BD Biosciences) as described previously (13 (link)).
5
Isolation and Characterization of Tumor-Infiltrating NK Cells
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The mouse tumors were freshly dissociated into single cells with the gentleMACS C tube by gentleMACS Dissociator (Miltenyi Biotec). Harvested cells were centrifuged at 1500 rpm for 8 min at 4 °C. Isolation of infiltrated cells were filtered through cell strainer (40 μm; BD Falcon) and counted by using 0.4% Trypan Blue Dye. The isolated cells were washed and blocked with 2% BSA-PBS (pH 7.4) and cells (2 × 105/tube) were incubated with immune cell markers for 30 min at 4 °C in the dark. For gating of NK cells (CD45+/CD3-/CD49+), mixture of antibodies including FITC-conjugated anti-Mouse CD45 at 1:200 dilution [BD Pharmingen, 5553079)], PerCP-CyTM5.5 anti-Mouse CD3 at 1:100 dilution [BD Pharmingen, 560527] and PE-conjugated anti-Mouse CD49b (at 1:100 dilution) [BD Pharmingen, 561066] was incubated with tumor dissociated cells (after filtering through cell strainer) for 30 min at 4 °C in the dark. The stained cells were washed with 2% BSA–PBS. Cells were then suspended in 2% BSA–PBS buffer. Positive stained cells were acquired using a Guava easyCyte HT instrument (Millipore) or FACSCalibur (Beckton Dickinson) according to the manufacturer’s operation manual and analyzed using FlowJo software.
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