The viability and viable cell count of ESCs and EpiLCs were calculated using Trypan blue (0.4%, Thermo Fisher) on a Countess II FL Automated Cell Counter (Thermo Fisher). For BrdU incorporation studies, cells were permeabilized, fixed, and stained using the BrdU Flow Kit (PerCP-Cy™5.5 Mouse anti-BrdU, BD Biosciences) before analysis by flow cytometry on a BD Biosciences LSRII (UCLA BSCRC Flow Cytometry Core). Quantification of PGCLCs proliferation was performed using CellTrace™ Yellow (5μM, added at the induction, Thermo Fisher), which binds to intracellular amines after diffusing through cell membranes. The overall fluorescent signal, which gradually decreases as cell division occurs, reflects the number of cell divisions occurring and was measured on a BD Biosciences LSRII (UCLA BSCRC Flow Cytometry Core). The FlowJo software was used to calculate the percentage of induction, the number of cell divisions and generate the associated graphs (version 10, FlowJo, LLC).
Biosciences lsrii
Manufactured by BD
The BD Biosciences LSRII is a flow cytometry instrument designed for multiparameter analysis of cells and particles. It is capable of detecting and analyzing multiple fluorescent signals simultaneously, providing detailed information about the physical and chemical characteristics of the samples. The LSRII is a versatile and widely-used tool in various fields of research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Trypan blue, by Thermo Fisher Scientific (4 mentions)
Celltrace yellow, by Thermo Fisher Scientific (4 mentions)
Countess 2 fl automated cell counter, by Thermo Fisher Scientific (4 mentions)
Brdu flow kit, by BD (1 mentions)
Click it edu assay, by Thermo Fisher Scientific (1 mentions)
Cd11b m1 70, by BioLegend (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Annexin 5 pacific blue, by BioLegend (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
F4 80, by BioLegend (1 mentions)
Annexin 5 apc, by BD (1 mentions)
6 protocols using biosciences lsrii
1
Quantifying Pluripotent Cell Proliferation
2
Cell Viability, Proliferation, and Differentiation Assays
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The viability and viable cell count of ESCs and EpiLCs were calculated using Trypan blue (0.4%, Thermo Fisher) on a Countess II FL Automated Cell Counter (Thermo Fisher). For BrdU incorporation studies, cells were permeabilized, fixed, and stained using the BrdU Flow Kit (PerCP-Cy5.5 Mouse anti-BrdU, BD Biosciences) before analysis by flow cytometry on a BD Biosciences LSRII (UCLA BSCRC Flow Cytometry Core). Quantification of PGCLCs proliferation was performed using CellTrace Yellow (5 µM, added at the induction, Thermo Fisher), which binds to intracellular amines after diffusing through cell membranes. The overall fluorescent signal, which gradually decreases as cell division occurs, reflects the number of cell divisions occurring and was measured on a BD Biosciences LSRII (UCLA BSCRC Flow Cytometry Core). The FlowJo software was used to calculate the percentage of induction, the number of cell divisions and generate the associated graphs (version 10, FlowJo, LLC).
3
Quantification of ESC, EpiLC, and PGCLC Viability
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The viability and viable cell count of ESCs and EpiLCs was calculated using Trypan blue (0.4%, Thermo Fisher) on a Countess II FL Automated Cell Counter (Thermo Fisher). For BrdU incorporation studies, cells were permeabilized, fixed, and stained using the BrdU Flow Kit (PerCP-Cy™5.5 Mouse anti-BrdU, BD Biosciences) before analysis by flow cytometry on a BD Biosciences LSRII (UCLA BSCRC Flow Cytometry Core). Quantification of PGCLCs proliferation was performed using CellTrace™ Yellow (5μM, added at the induction, Thermo Fisher), which binds to intracellular amines after diffusing through cell membranes. The overall fluorescent signal, that gradually decreases as cell division occurs, reflects the number of cell divisions occurring and was measured on a BD Biosciences LSRII (UCLA BSCRC Flow Cytometry Core). The FlowJo software was used to calculate percent of induction, numbers of cell division and generate the associated graphs (version 10, FlowJo, LLC).
4
Quantifying Pluripotent Cell Proliferation
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The viability and viable cell count of ESCs and EpiLCs were calculated using Trypan blue (0.4%, Thermo Fisher) on a Countess II FL Automated Cell Counter (Thermo Fisher). For BrdU incorporation studies, cells were permeabilized, fixed, and stained using the BrdU Flow Kit (PerCP-Cy™5.5 Mouse anti-BrdU, BD Biosciences) before analysis by flow cytometry on a BD Biosciences LSRII (UCLA BSCRC Flow Cytometry Core). Quantification of PGCLCs proliferation was performed using CellTrace™ Yellow (5μM, added at the induction, Thermo Fisher), which binds to intracellular amines after diffusing through cell membranes. The overall fluorescent signal, which gradually decreases as cell division occurs, reflects the number of cell divisions occurring and was measured on a BD Biosciences LSRII (UCLA BSCRC Flow Cytometry Core). The FlowJo software was used to calculate the percentage of induction, the number of cell divisions and generate the associated graphs (version 10, FlowJo, LLC).
5
MAGE-A3 Antibody Proliferation Assay
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For EDU assays, A431 cells were seeded at 1.0 x 105 cells per well in 6-well plates and treated with MAGE-A3 antibody at a 1:500 dilution mixed with antibody transport solution (ATS), which contained PBS, 50% glycerol, 0.02% sodium azide PH 7.3 and 0.1% dimethylsulfoxide (DMSO). At 72 hours post-treatment, the Click-IT EDU assay was performed according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). Flow cytometry was performed using BD Biosciences LSR II (BD Biosciences, San Jose, CA), and data were analyzed using FloJo2 software (BD Biosciences, San Jose, CA).
6
Multiparametric Analysis of BMDM
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BMDMs were detached from culture dishes and incubated with annexin V-Pacific Blue (BioLegend) or annexin V-APC (BD biosciences) and Fixable Viability Dye eFluor 780 (eBiosicence) or 7-AAD (Invitrogen) for cell death analysis, with CM-H2DCFDA (Invitrogen) or CellROX deep red staining kit (Thermo Fisher Scientific) for analysis of reactive oxygen species, or with antibodies (CD11b [M1/70], and; F4/80 [BioLegend]) for differentiation analysis. Fluorescence was detected by flow cytometry (BD Biosciences LSR II), and data were analyzed using FlowJo software (Tree Star). Events were gated to exclude dead cells and debris, then gated on FITC wave length (CM-H2 DCFDA), APC (CellROX deep red), PE-Cy5 (F4/80), Pacific Blue (CDllb or annexin V), or APC-Cy7 (fixable viability dye) when compared to unstained control.
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