Flow cytometric analysis of bone marrow, peripheral blood cells or enzymatically digested esophagus was conducted using the following antibodies: anti-CD11b (R&D), anti–GR-1 (BD Bioscience), anti–Siglec-F (BD Bioscience), anti-CCR3 (BD Bioscience), anti–PIR-A/B (ebioscience), IgG2b (ebioscience), anti-CD45 (ebioscience) and anti-CD11c (BD Bioscience). Cell counts were conducted using 123count beads (ebioscience) according to the manufacturers’ instructions. In all experiments, at least 50,000 events were acquired by (FACSCalibur, BD Bioscience), and data were analyzed using the Kaluza (BeckmanCoulter) or FlowJo (TreeStar) softwares.
123count beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 123count beads are fluorescent-labeled polystyrene particles designed for use in flow cytometry applications. They provide a standardized sample for calibrating and monitoring the performance of flow cytometers.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (1 mentions)
Anti cd45, by Thermo Fisher Scientific (1 mentions)
Igg2b, by Thermo Fisher Scientific (1 mentions)
Anti cd11b, by R&D Systems (1 mentions)
Anti gr 1, by BD (1 mentions)
Anti siglecf, by BD (1 mentions)
Anti cd11c, by BD (1 mentions)
Anti ccr3, by BD (1 mentions)
Kaluza, by Beckman Coulter (1 mentions)
Fbs stain buffer, by BD (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Xe 2100 hematology analyzer, by Sysmex (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Cyan adp lx, by Beckman Coulter (1 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)
4 protocols using 123count beads
1
Multiparameter Flow Cytometry of Immune Cells
2
Leukocyte Viability Staining and Counting
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Leukocyte viability stain (detectable in near-IR channels) was added into each 100 μL cell suspension aliquot and incubated at 21 °C for 10 min. Three milliliter of FBS stain buffer (BD Biosciences) was added to each cell sample for washing and centrifuged at 300 x g for 5 min at 21 °C. The supernatant was aspirated, and cells were resuspended in a solution containing a final concentration of 1025 beads/μL (123count beads, eBioscience) and 1.6% PFA (Electron Microscopy Sciences). Following a 10-min incubation at 21 °C, viability was assessed by running the samples through a flow cytometer. Where indicated, additional cell counts were obtained using a Sysmex XE-2100 hematology analyzer according to manufacturer’s protocol.
3
Comprehensive FACS Analysis of Immune Cells
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For FACS analysis cells were stained for 20 minutes at 4 °C by the application of the respective antibodies: aCD4 PE (RM4-5), aCD8 PE-Cyanine7 (53-6.7), aCD3 APC (OKT3), aCD14 APC (61D3), aCD31 (WM-59) (all from eBioscience; Frankfurt am Main, Germany). For live dead discrimination propidium iodide (Molecular Probes, Eugene, Oregon) was used. MHC-I staining was performed for 45 minutes at 4 °C using Strept-Tactin APC backbone (IBA GmbH, Göttingen, Germany). Data were collected by flow cytometry on a CyAn ADP Lx (Beckman Coulter) and analyzed with FlowJo software (TreeStar). Absolute cell numbers were calculated using 123 count beads (Thermo Fisher Scientific, Waltham, USA).
4
CAR-T Cell Quantification in Mice
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Peripheral blood and spleens were harvested from treated mice. A total of 30 µl of peripheral blood was then incubated in an antibody panel (CD3/CD45.1/CD45.2/CAR/IL-15Rα) at 4°C for 30 min, then red blood cells were lysed and fixed with red blood cell (RBC) lysis/fixation solution (BioLegend), and finally 10 µl of 123count beads (Thermo) was added for flow analysis and calculating the absolute number of CAR-T cells (expressed as CAR-T count per 100 μl blood). The spleens were gently crushed through a 40 μm cell strainer, followed by addition of ACK lysing buffer (Gibco) to make a cell suspension, then part of the resultant splenocytes were incubated in an antibody panel (CD3/CD45.1/CD45.2/CAR) and washed with FACS buffer twice, and finally 10 µl of 123count beads was added for flow analysis and calculating the absolute number of CAR-T cells.
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