Flow cytometry was performed to study drug effects on apoptosis. H1, H2 and H3 cell lines were plated in 6-well plates (Nunc) with a concentration of 3 x 105 cells/well in 4 mL growth medium. After 24 h, CCT196969 was added to the wells in final concentrations of 1, 2 and 4 μM. Untreated cells were used as controls. 4 h before the collecting of cells, 4.08 μL of 30% H2O2, equivalent to a concentration of 10 μM, was added in one well to induce apoptosis and serve as an apoptotic control. After 72 h of incubation, growth medium was collected from the wells into tubes, and the cells were washed with phosphate buffered saline (PBS), trypsinated and added to the growth medium before centrifuging at 900 rpm for 5 min. After removal of the supernatant, cells from each sample were resuspended in 100 μL Annexin V binding buffer (Invitrogen), and 2 μL of Annexin V and propidium iodide (PI; AlexaFluor®488 Annexin v/dead cell apoptosis kit; Molecular Probes, Life Technologies, Waltham, MA, USA) were added to the samples. After 20 min incubation the samples were vortexed and analysed using a BD Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA). Fluorescence in the FITC-A and PE-A channels were gated to a two-parameter histogram, and analysed using FloJo software (Tree Star Inc., Ashland, OR, USA). The experiment was repeated 3 times for each cell line.
Flojo software
Manufactured by Tree Star
Sourced in United States
FloJo software is a computational analysis tool designed for the quantification and analysis of flow cytometry data. It provides a suite of tools for data visualization, compensation, gating, and statistical analysis of multiparametric flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (14 mentions)
Lsrii flow cytometer, by BD (11 mentions)
Facscanto 2, by BD (6 mentions)
Facsdiva software, by BD (5 mentions)
Cellquest software, by BD (4 mentions)
Facscanto, by BD (4 mentions)
Facscalibur flow cytometer, by BD (4 mentions)
Cytofix cytoperm kit, by BD (4 mentions)
Cyan flow cytometer, by BD (3 mentions)
Lsr 2 flow cytometer, by BD (3 mentions)
6 well plate, by Thermo Fisher Scientific (2 mentions)
Percoll, by GE Healthcare (2 mentions)
Alexafluor 647 conjugated mouse anti human cd47 clone b6h12, by BD (2 mentions)
Cd163, by BD (2 mentions)
Abs specific for human cd206, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Alexa fluor 546 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Facscelesta cell analyzer, by BD (2 mentions)
Rabbit anti human c1q, by Thermo Fisher Scientific (2 mentions)
96 protocols using flojo software
1
Apoptosis Induction and Flow Cytometry
2
Flow Cytometry Analysis of Human Macrophages
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Human macrophages were detached using ice-cold 1.5 mM EDTA in Hank’s balanced salt solution. Cells were washed with precooled staining buffer (PBS containing 1% BSA and 0.02% NaN3) and incubated on ice for 30 min with 4.8 mg/ml human IgG (Beriglobin P; CSL Behring, King of Prussia, PA) to prevent nonspecific binding of the mAbs to Fc receptors. Then, antibody-fluorochrome conjugates with appropriate isotype controls were added. Cells were incubated for 30 min on ice and then washed with the staining buffer. Samples were analyzed on a LSRII flow cytometer (BD Biosciences) and the data were further processed with the FloJo software (Treestar, Ashland, OR). Living cells were gated according to their forward- and side-scatter characteristics and dead cells were excluded using DAPI or 7-aminoactinomycin D (Sigma-Aldrich). Cells were scored positively that had a higher fluorescence than the cut-off of 0.5% of the isotype control mAbs. In graphs, geometric mean of fluorescence corrected for background staining using matched isotype control mAb is shown.
3
Apoptosis Analysis in Multiple Myeloma
4
Immune Cell Isolation and Analysis
5
Quantifying DNA Damage Response by Flow Cytometry
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Cells were seeded in 90 mm dishes and left to attach overnight prior to treatment as indicated. Cells were then fixed in 70% methanol and stored overnight at −20°C. After washing in PBS, cells were resuspended in 2mLs PBS supplemented with 0.5% BSA (Sigma-Aldrich) and 0.25% Triton-X100. Following 15 min incubation on ice, cells were resuspended with the antibody, anti-γH2AX [(ser139) (#2577 Cell Signaling, MA, USA), 1:50] diluted in 50 μL of PBS supplemented with 0.5% BSA and 0.25% Triton-X100 and incubated for 2 hours. Cells were then washed with 0.25% Triton-X100 in PBS and incubated with the secondary antibody Alexfluor 488 goat anti-rabbit IgG (1:500, diluted in 100 μL PBS supplemented with 1% BSA) for 30 min protected from light. Following a final wash with PBS, cells were incubated with 5 μL RNaseA (2 mg/mL) and 200 μL propidium iodide (PI, 50 μg/mL) for 15 minutes in the dark. Samples were analysed by flow cytometry using the FACSCalibur 488 nm laser (BD Biosciences, CA, USA) and more than 10000 cells were counted. All experiments were repeated three times. Data were analysed using FLOJO software (Tree Star, San Carlos, CA, USA).
6
Immunophenotyping and Tetramer Staining
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Single cell suspensions were resuspended in PBS supplemented with 2% fetal bovine serum and 2mM EDTA. Samples were blocked with CD16/32 and stained using standard procedures, antibodies used for these studies are described in Table 1 . Events were recorded on BD LSRII flow cytometer and results were analyzed using FloJo software (Tree Star Inc). Tetramer staining was performed using MHV68 peptide specific tetramers, p79/H-2Kb and p56/H-2Db, generated by the NIH Tetramer Core at Yerkes National Primate Research Center at Emory University. For intracellular cytokine staining (ICCS) eBioscience fixable viability dye (catalog # 65-0865-14) was used.
7
Caspase Activation in CAR-T Cells
8
Intracellular Calcium Analysis of B Cells
9
Measuring Mitochondrial Membrane Potential in Flies
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Measurement of neural mitochondrial membrane potential from 30- to 35-day-old flies was conducted as previously described (Burman et al., 2012 (link)), except that dissections were performed in supplemented DME/Ham’s F-12 High Glucose media lacking phenol red (Sigma), and all incubation steps were carried out at 25°C. In addition, 10 nM tetramethylrhodamine ethyl ester (TMRE) was utilized throughout the protocol. The % of cells harboring polarized mitochondria was determined using FloJo software (Treestar Inc., Ashland, OR), and defined as the % of cells exhibiting relative TMRE fluorescence above 103 arbitrary fluorescence units, as previously described (Burman et al., 2012 (link)). All flow cytometry measurements were performed on a Becton-Dickinson LSR II flow cytometer.
10
Multiparametric Analysis of T Cell Responses
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Spleens were collected and tissue was processed into single-cell suspension. Surface staining was conducted by incubating splenocytes with appropriate antibody cocktails for 20 min at 4°C. Endogenous memory and naïve CD8 T cells were detected based upon surface staining with anti-CD8 (clone 53-6.7, eBioscience) and anti-CD11a (clone M17/4, eBioscience) as previously described (30 (link)). P14 cells were detected based upon surface staining with anti-CD8 and anti-Thy1.1 (clone His51, eBioscience), and in some instances 1° earlyM and lateM or 1° and 3° memory P14 cells were distinguished from one another based upon additional surface staining with anti-Thy1.2 (clone 53-2.1, eBioscience). Intracellular cytokine staining was performed using anti-IFN-γ (clone XMG1.2, eBioscience) or anti-granzymeB (anti-GrB; clone GB12, Invitrogen). Flow cytometry data was acquired using FACSCanto (BD Biosciences, San Jose, CA, USA) and analyzed using FloJo software (Tree Star Inc., Ashland, OR, USA).
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