Tumorspheres were dissociated and single cells resuspended in PBS. Samples were sorted using a MoFlo XDP Cell Sorter (Beckman Coulter). Dead cells were excluded using the viability dye 7AAD (1:10, Beckman Coulter) or using a near IR Live/Dead fixable staining kit (Life Technologies). Compensation was performed using mouse IgG CompBeads (BD). GFP expression was defined as positive or negative based on the analysis of regions established by the isotype control. Cells were sorted into tubes containing 1 mL BTIC enrichment medium and small aliquots from each sort tube were analyzed to determine the purity of the sorted populations. Cells were allowed to equilibrate at 37 °C for a few hours prior to experimentation. Intracellular levels of Bmi1 and Sox2 were determined following preparation using Fixation/Permeabilization Solution Kit (Cat # 554714, BD Biosciences) along with antibodies anti-Bmi1 (1:11; Cat # 130-106-736, Miltenyi) and anti-Sox2 (1:20; Cat # 561610, BD Horizons), for which viable cells were stained using LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Cat # L10119, Thermo Scientific).
Mouse igg compbeads
Manufactured by BD
Sourced in United States
Mouse IgG CompBeads are a set of polystyrene beads coated with mouse immunoglobulin G (IgG) that can be used as a compensation control in flow cytometry experiments. These beads are designed to match the scatter and fluorescence properties of mouse IgG-stained cells, allowing for accurate compensation adjustment.
Automatically generated - may contain errors
Lab products found in correlation
Moflo xdp cell sorter, by Beckman Coulter (5 mentions)
7 aad, by Beckman Coulter (2 mentions)
Apc conjugated anti cd133, by Miltenyi Biotec (2 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Anti cd3, by BD (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Liberase, by Roche (1 mentions)
Goat anti human apc fab igg, by Jackson ImmunoResearch (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Liberase blendzyme 3, by Merck Group (1 mentions)
Dnase, by Worthington (1 mentions)
5 protocols using mouse igg compbeads
1
Isolation and Analysis of Tumor-Initiating Cells
2
GBM and CAR-T Cell Immunophenotyping
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GBM cells and T cells in single cell suspensions were resuspended in PBS+2 mM EDTA. GBM cells were stained with he_L or he_Lm IgG (0.064–1000 nM) or by IgG control AffiniPure Goat Anti-Human IgG, F(ab’)2 fragment specific, Jackson ImmunoResearch, Cat#109-005-006) or APC-conjugated anti-CD70 antibody (Miltenyi Biotech, REA 292) and incubated for 30 min on ice. CAR-T cells were stained with fluorescent tagged anti-CD3 (BD Biosciences, Cat#557851), anti-NGFR (Miltenyi Biotech, Cat#130-112-790) and anti-c-Myc (Miltenyi Biotech, Cat#130-116-653). Samples were run on a MoFlo XDP Cell Sorter (Beckman Coulter). Dead cells were excluded using the viability dye 7AAD (1:10; Beckman Coulter, A07704). Compensation was performed using mouse IgG CompBeads (BD Biosciences, Cat#552843).
3
Isolation of CD133+ Cancer Stem Cells
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Tumorspheres were suspended in 1 mL of PBS, dissociated into single cells using Liberase Blendzyme 3, washed with PBS, resuspended in PBS + 2 mM EDTA and filtered through 35 μm filter. Single-cell suspensions were stained with APC-conjugated anti-CD133 or a matched isotype control (Miltenyi) as recommended by the manufacturer and incubated for 15 min at room temperature. Samples were run on a MoFlo XDP Cell Sorter (Beckman Coulter). Dead cells were excluded using the viability dye 7AAD (1%; Beckman Coulter). Compensation was performed using mouse IgG CompBeads (BD Biosciences). Expression of CD133 was defined as positive or negative based on the analysis regions set on the isotype control.
4
CD133+ Cell Isolation and Sorting
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Tumorspheres/cell aggregates were dissociated using liberase (Roche, Laval, QC, Canada) and cells at a concentration of 1 million single cells/ml in phosphate-buffered saline+2 mM EDTA were stained with APC-conjugated anti-CD133 or a matched isotype control (Miltenyi, Auburn, CA, USA). The MoFlo XDP Cell Sorter was used to analyze/sort samples and Kaluza software (Beckman Coulter, Mississauga, ON, Canada) was used for analysis. Mouse IgG CompBeads (BD, San Jose, CA, USA) were used for compensation and viability dye 7-AAD dye (1:10; Beckman Coulter) was added in samples to exclude dead cells. Purity check for CD133 expression levels after sorting was also performed.
5
CA9 Surface Expression Analysis
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GBM Tumorspheres were dissociated using 0.2 Wünsch unit/mL Liberase Blendzyme 3 (Millipore Sigma, Cat#5401119001) plus 10 μL DNase (Worthington Biochemical, Cat#LK003170) and adherent cultures were dissociated using dissociation enzyme TrypLE (ThermoFisher, Cat#12605028). The single cells were resuspended in PBS+2 mM EDTA (Invitrogen, Cat# AM9260G). Cells were then stained with APC conjugated mouse monoclonal human Carbonic Anhydrase 9 antibody (1:10) (R&D, Cat#FAB2188A) or a matched isotype control and CA9 DATEs followed by goat anti human APC-Fab IgG (1:2000, Jackson ImmunoResearch, Cat#109-136-170) and incubated for 15 minutes at room temperature. T cells were stained with CA9 DATEs (15 minutes RT) followed by goat anti human APC-Fab IgG (1:2000, Jackson ImmunoResearch, Cat#109-136-170), anti-CD25 (Miltenyi Biotech, Cat#130-113-283) and anti-CD69 (BD Bioscienecs, Cat#555533). Samples were run on a MoFlo XDP Cell Sorter (Beckman Coulter). Dead cells were excluded using the viability dye 7AAD (1:10; Beckman Coulter, Cat#A07704). Compensation was performed using mouse IgG CompBeads (BD Biosciences, Cat#552843). Samples were run on a MoFlo XDP Cell Sorter (Beckman Coulter) to assess the level of CA9 surface expression.
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