Cells were plated on 24-multiwell plates, then fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BD), in accordance with the manufacturer’s instructions. Next, antibodies diluted in Perm/Wash buffer (BD) were added and then samples were incubated with antibodies overnight at 4°C. Nuclei were counter-stained with Hoechst 33342 (10 μg/ml, Sigma) for 15 min at room temperature. Immunofluorescence images were obtained using a fluorescence microscope (Leica) and confocal images were obtained using a confocal microscope (Zeiss). For staining, Oct3/4 (AlexaFluor488, 1:50, BD Pharmingen), Nanog (PE, 1:50, BD Pharmingen), Sox2 (AlexaFluor555, 1:50, BD Bioscience), Tra-1-60 (AlexaFluor488, 1:100, BD Pharmingen), SSEA-3 (PE, 1:100, BD Pharmingen), SSEA-4 (AlexaFluor488, 1:100, BD Bioscience), Tuj1 (AlexaFluor488, 1:100, BD Bioscience), Nestin (AlexaFluor488, 1:100, Millipore), CD107a (APC, 1:100, Biolegend), HLA-G (PE, 1:100, Abcam), HLA-BC (APC, 1:100, eBioscience), human β2m (Abcam), and goat anti-mouse IgG secondary (AlexaFluor555, Thermo Fisher) antibodies were used.
Alexa fluor 488
Manufactured by BD
Sourced in United States
Alexa Fluor 488 is a fluorescent dye used in various laboratory applications, such as flow cytometry, immunohistochemistry, and fluorescence microscopy. It has an excitation maximum of approximately 495 nanometers and an emission maximum of around 519 nanometers, producing a bright green fluorescence. Alexa Fluor 488 is commonly used for labeling proteins, cells, and other biological molecules.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (6 mentions)
Alexa fluor 647, by BD (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fluorescein isothiocyanate (fitc), by BD (2 mentions)
Pe cyanine7, by BD (2 mentions)
Facs diva high speed cell sorter, by BD (2 mentions)
Anti stro 1, by Thermo Fisher Scientific (2 mentions)
Facsaria 3, by BD (2 mentions)
Anti cd90, by BD (2 mentions)
Anti cd105, by BD (2 mentions)
Anti cd45, by BD (2 mentions)
Anti stro 1, by BD (2 mentions)
Percp cy5, by BD (2 mentions)
Alexa fluor 594, by BD (2 mentions)
Goat anti rabbit igg, by BD (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Igg2a pe, by BD (1 mentions)
Pecy7 anti cd45, by BD (1 mentions)
45 protocols using alexa fluor 488
1
Immunofluorescence Analysis of Stem Cell Markers
2
Immunophenotyping of Mouse Immune Cells
3
Multiparametric Flow Cytometry of Neural Stem Cells
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The dissociated neurospheres were examined by FACSCalibur flow cytometry (BD Instruments Inc., USA) for detection of following markers: β-tubulin III (Alexa Fluor 488), anti-SOX-2 (PE Mouse), anti-nestin (Alexa Fluor 647) — all acquired from Becton Dickinson (BD) — anti-GFAP (Glial fibrillary acidic protein, Sigma-Aldrich) and anti-CD133 (anti-PROM-1, Abnova).
The single-cell suspensions (0.5x106 cell/tube) were washed, fixed, for 15 min at RT, with 2% formaldehyde in PBS and permeabilized for 5 min with Triton X-100 at RT for intracellular markers. Cells were washed with PBS and then incubated, for 1h, at RT, with selected primary antibodies for 1h at RT in the dark. For CD133 and GFAP detection, secondary PE (Abcam) and FITC-conjugated (Millipore) antibodies respectively were used for 1h at RT. The population of interest was gating according to its Forward Scatter (FSC)/Side Scatter (SSC) criteria. 10,000 events were acquired for each sample and analyzed by CellQuest software (BD Instruments Inc., USA).
The single-cell suspensions (0.5x106 cell/tube) were washed, fixed, for 15 min at RT, with 2% formaldehyde in PBS and permeabilized for 5 min with Triton X-100 at RT for intracellular markers. Cells were washed with PBS and then incubated, for 1h, at RT, with selected primary antibodies for 1h at RT in the dark. For CD133 and GFAP detection, secondary PE (Abcam) and FITC-conjugated (Millipore) antibodies respectively were used for 1h at RT. The population of interest was gating according to its Forward Scatter (FSC)/Side Scatter (SSC) criteria. 10,000 events were acquired for each sample and analyzed by CellQuest software (BD Instruments Inc., USA).
4
Phagocytosis of Francisella tularensis
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A20 cells or peritoneal cells (5 x 105 per well) were co-cultivated with unopsonized or opsonized F. tularensis LVS, F. tularensis LVS/GFP, or F. tularensis mutants, in total volume of 0.5 mL per well at multiplicity of infection (MOI) 500. Control A20 cells or peritoneal cells were cultivated without infection. After 3 h incubation at 36.8°C in 5% CO2 atmosphere, cultures were washed using PBS. The proportion of infected cells with F. tularensis LVS/GFP was examined using flow cytometry. F. tularensis was stained using rabbit anti-F. tularensis serum as a primary antibody and goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Becton Dickinson, San Jose, California, USA) as a secondary antibody for fluorescent microscopy. DAPI (Invitrogen, Molecular Probes, Oregon, USA) was used to visualize the nuclei.
5
Acute Myeloid Leukemia Cell Differentiation
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Morphologic differentiation of OCI-AML3, IMS-M2, K562 or patient cells was determined by CD11b expression and morphologic assessment of cells stained with May-Grünwald-Giemsa. Cells were treated for 48 h and 72 h with either 500 nM OTX015 (MK-8628), 500 nM JQ1, 1 µM ATRA + 1 µM ATO, or 0.1% DMSO followed by flow cytometric detection of the differentiation marker CD11b Alexa Fluor® 488 (#557701, Becton Dickinson, Le Pont de Claix, France) using a CytoFLEX flow cytometer (Beckman Coulter, Villepinte, France) and analyzed with the FlowJo flow cytometry software (FlowJo LLC, Ashland, OR, USA). For morphologic analysis, cytospin preparations (Cytospin 4, Fisher Scientific, Illkirch, France) of OCI-AML3, IMS-M2, K562 or patient cells treated for 48 h by OTX015 (MK-8628) 500 nM, JQ1 500 nM, ATRA 1 µM + ATO 1 µM, or 0.1% DMSO were stained with May-Grünwald Giemsa solution and examined under light microscopy with a Zeiss microscope (Carl Zeiss, Marly le Roi, France) with a X40 objective.
6
Immunofluorescent Analysis of β-Catenin in Colon Tissue
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Slides of colon tissue (5 μm) were deparaffinized with xylene and rehydrated with decreasing gradient alcohol. Antigen retrieval was carried out by pressure‐cooking slides for 3 min in 0.01 M citrate buffer (pH 6.0). To avoid non‐specific interactions of antibodies, the slides were treated for 2 h in 5% BSA in PBS. Immunostaining was performed by incubation overnight with anti‐β‐catenin (1:100, Alexa Fluor® 488, Cat. 562505, BD Pharmingen™) at 4°C. The slides were mounted on microscope slides using Mowiol + 4′,6‐diamidino‐2‐phenylindole (DAPI) for nuclear staining. The images were acquired at room temperature and detected under 63× magnification (numerical aperture of the objective lens: 1,4), by using a laser‐scanning confocal microscope with AiryScan2 module (Zeiss, LSM 900 associated) (Vanacore et al., 2018 (link)). Three fields for each colon were analysed by Zeiss Zen blue edition software.
7
Pluripotency Assessment of Human iPSCs
8
Apoptosis Assay for Activated PBMCs
9
Immunophenotyping of iPSC-derived SKPs
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After cells were detached and collected, they were fixed and permeabilized with cytofix buffer for 20 min. After washing with Phosflow Perm/Wash buffer, cells were incubated for 30 min with the anti-nestin antibody conjugated with PerCP-CyTM5.5 and with the anti-fibronectin antibody conjugated with Alexa fluor 488 (both from BD Biosciences). Isotype-matched monoclonal antibodies (mAbs) were used as negative controls. In order to calculate the differentiation efficiency of iPSCs into SKPs, stained cells were analyzed using BD FACSVerse with FACSuite software (BD Biosciences)
10
Cytarabine and AMD070 Combination Therapy for NALM-6 Leukemia
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