Live cells were incubated with conjugated antibodies CD133-PE (1:100; 12-1338, eBioscience, San Diego, CA, USA) and CD44-FITC (1:50; ab19622, abcam, Cambridge, UK) at 4 °C for 30 min. PE Mouse IgG1 kapa (400111, Biolegend) and FITC Rat IgG2b (400633, Biolegend) were used as isotype controls. After washing with PBS, cells were re-suspended in 300 μl PBS and examined on an LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). For calcium detection, live cells were loaded with 5 μM Fluo3-AM (Sigma) for 45 min at 37 °C, washed with PBS, and checked on the flow cytometer. For cell cycle analysis, transfected cells were dissociated into single cell suspension and fixed in 70% ethanol. After washing with PBS, cells were treated with RNase (0.2 mg/ml) and stained in propidium iodide solution (10 μg/ml) and then analyzed by flow cytometry.
Fitc rat igg2b
Manufactured by BioLegend
FITC Rat IgG2b is a laboratory reagent used in flow cytometry applications. It is a fluorescently labeled immunoglobulin G (IgG) antibody derived from the rat. The FITC (fluorescein isothiocyanate) fluorophore is conjugated to the IgG2b antibody, allowing for the detection and analysis of target cells or molecules.
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Lab products found in correlation
Lsrii flow cytometer, by BD (1 mentions)
Cd133 pe, by Thermo Fisher Scientific (1 mentions)
Cd44 fitc, by Abcam (1 mentions)
Fluo 3 am, by Merck Group (1 mentions)
Bx63 microscope, by Olympus (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Cellsens software, by Olympus (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
2 protocols using fitc rat igg2b
1
Multiparameter Flow Cytometry Analysis
2
Immunofluorescent Staining of Mouse Thymus
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Seven-week old mice were killed by cervical dislocation. Thymuses were removed and snap frozen in CO2 snow. Ten µm thick sections were fixed in 4% PFA, blocked for 1h with PBS, 0.2 % Triton X-100, 3% BSA, stained for 1h with rat anti-mouse PDCA-1 Alexa Fluor® 488 (927; Biolegend) with either purified rabbit anti-mouse keratin 14 (Covance) or purified rabbit anti-mouse laminin (Cedarlane) antibodies and stained for an additional hour with donkey anti-rat IgG A488 (Molecular Probes; Invitrogen) and donkey anti-rabbit IgG A555 antibodies (Molecular Probes; Invitrogen). The sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen) and mounted with Fluorescence Mounting Medium (Dako). Control sections incubated with an isotype-matched primary antibody (FITC rat IgG2b,κ Biolegend)) or a "negative control rabbit immunoglobulin fraction" (DAKO) showed no staining. The sections were visualized with an Olympus DP71 digital camera mounted on an Olympus BX51 microscope (Olympus) or with Olympus DP80 digital camera mounted on an Olympus BX63 microscope (Olympus). Images were acquired with the Cellsens software (Olympus) and the images were merged using Image J (National Institutes of Health).
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