Spleen and thymus were collected from WT, Dnmt3a1−/− or Dnmt3a2−/− mice at P21, and single cell suspensions were generated by trituration and filtration through 40-μm nylon cell strainers (Corning Falcon). Cells were stained with fluorochrome-labeled antibodies at room temperature for 20 min, washed and analyzed on an LSR II flow cytometer (BD). All antibodies used in flow cytometry were purchased from BD or Thermo Fisher Scientific (eBioscience) and used at 1:100 dilution: anti-CD4, eFluor 450 (48-0042-82); anti-CD8a, eFluor 450 (48-0081-82); anti-CD45R, eFluor 450 (48-0452-82); anti-CD11b, PE-Cy7 (25-0112-82); anti-Gr-1, PE-Cy7 (25-5931-82); anti-CD45R, PE-Cy7 (25-0452-82); anti-CD4, FITC (11-0041-85); anti-CD8a, PE (BD, 553033). Flow cytometry data were processed using FlowJo software (v10.7.1).
Efluor 450
Manufactured by BD
Sourced in United States
EFluor 450 is a fluorescent dye that can be used for flow cytometry applications. It is designed to emit a fluorescent signal in the blue-violet spectrum, with a peak excitation wavelength of 450 nm and a peak emission wavelength of 520 nm. The dye can be used to label and detect various cellular components or molecules of interest.
Automatically generated - may contain errors
Lab products found in correlation
Efluor 450, by Thermo Fisher Scientific (3 mentions)
Lsrii flow cytometer, by BD (2 mentions)
C kit apc eflour780 2b8, by BD (2 mentions)
Cd150 alexa fluor 647 tc15 12ff12, by BD (2 mentions)
Sca 1 pe cy7 d7, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Anti cd8a, by BD (1 mentions)
40 μm nylon cell strainer, by Corning (1 mentions)
Anti cd4, by Thermo Fisher Scientific (1 mentions)
Anti cd45r, by BD (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Percoll gradient, by Merck Group (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Anti cd11b percp cy5, by BD (1 mentions)
Eflour450, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Clone cd28, by Beckman Coulter (1 mentions)
Clone okt3, by Thermo Fisher Scientific (1 mentions)
9 protocols using efluor 450
1
Dnmt3a knockout immune cell analysis
2
Multiparameter FACS Analysis of Hematopoietic Stem Cells
3
Phagocytosis of Tumor Cells by Macrophages
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Antibody dependent phagocytosis was performed as described in [40 (link)]. In short, macrophages were seeded in the presence of M-CSF for 2 days prior to the experiment in 24 wells plates (200,000/well). At day 0 B16F10-gp75 were harvested and stained with cell proliferation dye eFluor 450 (eBioscience) according to manufacturer’s protocol. Both antibodies (1 µg/mL) and tumor cells (E:T = 15:1) were diluted in complete medium. After 24 h of co-culture at 37 °C, cells were collected with Trypsin/EDTA and scratching. Subsequently samples were blocked with human serum, stained with anti-HLA-DR to stain macrophages, and fixed with 4% paraformaldehyde in PBS. Data was acquired and analyzed with the BD LSRFortessa X-20 and Flowjo X. The percentage of double-positive (HLA-DR+, eFluor 450+) macrophages was determined.
4
In Vitro T Reg Suppression Assay
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Using humanized mice, the functional activity of Tregs expanded in vivo by IgG-(IL-2N88D)2 was determined in an in vitro co-culture assay measuring the ability of Tregs to suppress the proliferation of CD4+ memory effector T cells stimulated by anti-CD3 and anti-CD28. Mice were treated with IgG-(IL-2N88D)2 and spleen cells were sorted into Tregs (CD45+CD3+CD4+CD25+CD127lo) and memory effector T cells (CD45+CD45RA−CD3+CD4+CD25+/-CD127hi). Prior to sorting, the effector T cells were stained with the cell proliferation dye eFluor 450 (eBioscience) according to the manufacturer's instructions. Memory effector T cells were cultured for three days at 5 × 104 cells in U-bottom plates and stimulated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (2 μg/ml); Tregs were added to the effector cells at a 1:1 ratio. Effector T cells stained with proliferation dye eFluor 450 were assessed for cell division using flow cytometry on a LSRFortessa (BD Biosciences) and analyzed with FlowJo software.
5
Flow Cytometric Analysis of Intestinal Macrophages
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Cells were isolated from the jejunum as described above. Cellular debris was removed from lamina propria mononuclear cells using Percoll gradient (Sigma-Aldrich). Antibodies for flow cytometry were purchased from eBioscience (Waltham, MA, USA) unless otherwise stated. Dead cells were excluded from analysis with a viability dye eFluor 780. Macrophages in the lamina propria were identified through staining with anti-major histocompatibility complex (MHC) class II (eFluor 450), anti-CD11b (PerCP/Cy5.5), and anti-F4/80 (BV605; BD Biosciences, San Diego, CA, USA) antibody. We also performed staining with anti-CHI3L1 (PE; Biorbyt, Cambridge, UK) antibody to determine if CHI3L1 is expressed in lamina propria macrophages. Stained cells were detected by flow cytometry using an LSR Fortessa™ X-20 cell analyzer (BD Biosciences), and data were analyzed using FlowJo software (version 10.6.0; Tree Star, Inc., Ashland, OR, USA). The gating strategy is shown in Supplementary Fig. S1 .
6
Multicolor Flow Cytometry Analysis
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Surface levels of CD4, BST-2, CCR7 and NTB-A were assessed by staining cells with their appropriate antibodies at 4°C for 30 min in buffer (1 × PBS + 3% FBS). An additional step including a secondary antibody was necessary to detect BST-2 surface levels. A viability dye, eFlour 450 (eBioscience) was then used to distinguish live from dead cells. Fixation was achieved using 0.5% Paraformaldehyde (PFA).
In experiments involving surface analysis of CD4 and detection of intracellular p24, cells were first probed with anti-APC-CD4, stained with eFluor 450, permeabilized (Cytofix/Cytoperm: BD Biosciences) and then stained with mouse-anti-FITC-p24. Total levels of CD4 in primary CD4+ T cells were measured by staining cells with eFlour 450, permeabilization and then probing with anti-APC-CD4. All data was collected on a BD FACS CantoII and analyzed with FlowJo software.
In experiments involving surface analysis of CD4 and detection of intracellular p24, cells were first probed with anti-APC-CD4, stained with eFluor 450, permeabilized (Cytofix/Cytoperm: BD Biosciences) and then stained with mouse-anti-FITC-p24. Total levels of CD4 in primary CD4+ T cells were measured by staining cells with eFlour 450, permeabilization and then probing with anti-APC-CD4. All data was collected on a BD FACS CantoII and analyzed with FlowJo software.
7
Mye-EUNITER T Cell Proliferation Assay
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The assay was performed according to the standardized Mye-EUNITER protocol as described.30 (link) Briefly, allogenic to monocytes CD3 T cells were isolated from healthy donor PBMC by MACS (Milteny Biotec) and labeled with 10 µM cell proliferation dye eFluor 450 (eBioscience) at RT for 20 min followed by the co-incubation for 4 days with monocytes prestimulated for 24 hours with rHSP90a (2 µg/mL). Cells were co-cultured in 96-well round-bottom plates (Sarstedt) precoated for 2 hours with anti-CD3 (1 µg/mL, clone OKT-3, eBioscience) and anti-CD28 antibodies (2 µg/mL, clone CD28.2, Beckman Coulter). T cell proliferation was measured by assessing proliferation dye eFluor 450 dilution by FACS-Lyric (BD Biosciences) flow cytometer.
8
Multicolor Flow Cytometry Analysis
9
Multiparameter FACS Analysis of Hematopoietic Stem Cells
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Multiparameter FACS analyses were performed to determine populations of HSCs (Lineage−Sca-1+c-Kit+Flk2−CD48−CD150+), multipotent progenitor sub-population 1 (MPP1, Lineage−Sca-1+c-Kit+Flk2−CD48−CD150−), MPP2 (Lineage−Sca-1+c-Kit+Flk2−CD48+CD150+), and MPP3 (Lineage−Sca-1+c-Kit+Flk2−CD48+CD150−). Antibodies were purchased from eBiosciences, San Diego, CA, unless otherwise noted. BM cells freshly harvested from femurs and tibias were stained with biotin-labeled antibodies against mouse hematopoietic lineage markers: Mac-1 (M1/70), Gr-1 (RB6-8C5), Ter119 (TER-119), CD3e (145-2C11), B220 (RA3-6B2), and subsequently stained with antibodies conjugated with various fluorochromes: streptavidin eFluor® 450, Sca-1-PE-Cy7 (D7, BD Biosciences, San Jose, CA), c-Kit-APC-eFlour780 (2B8), CD150-Alexa Fluor® 647 (TC15-12FF12.2, BD Biosciences), CD48-FITC (HM48-1), Flk2-PE (A2F10.1, BD Biosciences) for HSCs or MPPs. Specific cell populations were gated based on immune phenotypes for quantification or cell sorting. Fluorescence Minus One (FMO) was used for setting the gating on control samples. Flow cytometric analyses were performed on BD LSR II (BD Biosciences, San Jose). Cell sorting was conducted using BD FACSAria. Data were analyzed using FlowJo software (TreeStar, Ashland).
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