The percentage of Ki67-mediated cell proliferation as a function of local and global cell density was measured by a staining method using the Ki67 protocol using an Anti-human Ki67 FITC, Flow Cytometry Staining Buffer, and Foxp3 Fixation / Permeabilization solution (eBioscience) according to the manufacturer’s instructions. OVCAR-8 were seeded in 150 mm dishes in drug-free medium and incubated at 37° for 24hr to allow cells to attach. Three different global cell densities (7%, 20%, 33%) were seeded, each density in two different geometrical configurations. While the random formation was mixed very well, the circular formation was 15 dots containing 2/3 × 105, 2 × 105, and 10/3 × 105 cells per dot, respectively. The percentages of proliferation were then evaluated by FACS (BD LSRFortessa) every day for 4 days. Data from 10,000 gated events per sample were collected. Data were collected three times. For experiments with drug, after 24 hr with drug-free media, random and circular plates from each density were dosed with IC50 paclitaxel, 0.001 ± 6.20E-6 μM/L, and with IC50 × 4 paclitaxel. After 3 days of incubation with the drug, data were collected. See Table S3 for the results.
Foxp3 fixation permeabilization solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Foxp3 Fixation/Permeabilization solution is a laboratory reagent used for the fixation and permeabilization of cells prior to intracellular staining and flow cytometry analysis. The solution is designed to allow for the detection of intracellular proteins, such as the transcription factor Foxp3, which is commonly used as a marker for regulatory T cells.
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Lab products found in correlation
Novocyte, by Agilent Technologies (3 mentions)
Anti c myc, by Cell Signaling Technology (2 mentions)
Mitoview green mvg, by Biotium (2 mentions)
Mitospy orange, by BioLegend (2 mentions)
Anti phospho p70s6k thr421 ser424, by Cell Signaling Technology (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Permeabilization solution, by Thermo Fisher Scientific (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Flow cytometry staining buffer, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Rabbit anti aat, by Abcam (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Facscalibur, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Human fc block, by BD (1 mentions)
Anti cd4 pe a161a1, by BioLegend (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
14 protocols using foxp3 fixation permeabilization solution
1
Density-dependent Ki67 Proliferation
2
Metabolic Profiling of CD4+ T Cells
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After culture, CD4+ T cells (0.2 × 106) were stained with metabolic probes diluted in T-cell culture media for 20 min at 37° celsius. Cells were then washed twice in staining buffer (PBS/2% FCS) and resuspended before analysis. Metabolic probes used included MitoSpy Orange (MSO) (25 nM; BioLegend, Cat# 424803), and MitoView Green (MVG) (50 nM; Biotium, Cat# 70054). For analysis of c-Myc expression and p70S6K phosphorylation, cells (0.2 × 106) were first fixed in FoxP3 fixation/permeabilization solution (eBioscience, Cat# 005523–00) for 20 min at 37° celsius, washed in FoxP3 permeabilization buffer and incubated with anti-c-Myc (Cell Signalling Technology, Cat# 5605T) or anti-phospho p70S6K (Thr421/ Ser424) (Cell Signalling Technology, Cat# 9204S) for 30 min at 4° celsius. After washing, cells were then incubated with secondary antibody anti-Rabbit IgG (H + L), AF555 (Invitrogen, Cat# A-21428) for 20 min at 4° celsius, followed by a final wash. Samples were run on the BD LSRFortessa X-20, data collected using BD FACSDiva Software, and analysed using FlowJo version 10.7.1 (BD Biosciences).
3
Analyzing Surface Markers and Intracellular Cytokines
4
Metabolic Profiling of CD4+ T Cells
5
Characterizing Pluripotency in hESCs
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The hESC were dispersed into single cells by TrypLE (Invitrogen) treatment for 7 min at 37 °C, fixed in a Foxp3 Fixation/Permeabilization Solution (eBioscience) for 1 h on ice, and incubated in 5% (v/v) donkey serum for 15 min to reduce any nonspecific binding of antibodies. Cell were then exposed to an antibody directed against POU5F1 (1:200, Santa Cruz Biotechnology, catalog no. sc-9081) or to IgG (0.4 μg/mL; Santa Cruz Biotechnology, catalog no. sc-66931) in the blocking buffer for 1 h. All the steps were performed in the dark on ice, and cells were washed by Permeabilization Solution (eBioscience) three times between each step. For each cell population, at least 10,000 cells were analyzed in the Accuri C6 Flow Cytometer (BD Biosciences). Data were analyzed by the FlowJo (version X) software.
6
Multiparameter Flow Cytometry Analysis
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For the analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA and the appropriate antibodies. For intracellular cytokine staining, 5 x 105 naive CD8+ T cells were activated and cultured in 0.5 ml complete RPMI medium for 3–4 d, followed by restimulation by direct addition of stimulation cocktails of PMA, ionomycin, brefeldin A and monensin (500×) into the medium and then were incubated for 4 h. After incubation, cells were washed twice with PBS and resuspended in PBS containing 2% (wt/vol) BSA. Cells were then fixed and permeabilized using Foxp3 Fixation/Permeabilization solution according to the manufacturer’s instructions (eBioscience). Cells were then stained with the indicated antibodies listed in Supplementary Table 1 and the Reporting Summary. Flow cytometry data were acquired on Novocyte (ACEA Biosciences) or LSRII (Becton Dickinson) and were analysed with FlowJo software (TreeStar), and gating strategies are shown in Supplementary Fig. 3 .
7
Liver Cell Isolation and Characterization
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Cells were dissociated by incubation with 0.05% collagenase IV (Invitrogen) at 37°C for 15 minutes followed by incubation with Accutase (Innovative Cell Technologies, San Diego, CA, USA) at 37°C for 15 minutes. Dissociated cells were fixed and permeabilized with Foxp3 Fixation/Permeabilization solution (eBioscience, San Diego, CA, USA) for 1 hour at room temperature (RT). One microgram of mouse anti-ALB (R&D Systems) and rabbit anti-AAT (Abcam, Cambridge, UK) was conjugated using the Zenon R-Phycoerythrin Mouse IgG2a Labeling Kit and the Zenon Alexa Fluor 488 Rabbit IgG Labeling Kit (Invitrogen), respectively, according to the manufacturer’s instructions. Cells were incubated at RT for 1 hour with each labeled antibody. Cells were also labeled with the isotype control as a negative control. Flow cytometry was performed using BD FACS Calibur (BD Biosciences).
8
Comprehensive Immune Cell Analysis
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For analysis of surface markers, cells were stained in PBS containing 2% (w/v) BSA and the appropriate antibodies from Biolegend. Foxp3 expression was performed using the Foxp3 staining kit from eBioscience. For intracellular cytokine IFN-ɣ and IL-17A staining, T cells were stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin before being stained with CD4 antibody. Cells were then fixed and permeabilized using Foxp3 Fixation/Permeabilization solution according to the manufacturer's instructions (eBioscience™).
Cell total protein level was assessed by intracellular FITC (Fisher scientific) staining. Cell proliferation was assessed by CFSE staining per the manufacturer's instructions (Invitrogen). Cell viability was assessed by 7-AAD staining per the manufacturer's instructions (Biolegend). Flow cytometry data were acquired on Novocyte (ACEA Biosciences) and were analyzed with FlowJo software (TreeStar).
Cell total protein level was assessed by intracellular FITC (Fisher scientific) staining. Cell proliferation was assessed by CFSE staining per the manufacturer's instructions (Invitrogen). Cell viability was assessed by 7-AAD staining per the manufacturer's instructions (Biolegend). Flow cytometry data were acquired on Novocyte (ACEA Biosciences) and were analyzed with FlowJo software (TreeStar).
9
Isolation and Co-culture of B and T Cells
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CD19+ B lymphocytes and CD4+ CD25−/int CD127+ conventional T-helper (Tcon) lymphocytes were isolated from peripheral blood of melanoma patients using the BD FACS Aria II cell sorter. Purified B and Tcon lymphocyte suspensions were co-cultured (1x105 each/well) in sterile DMEM (10% FBS, 50U/ml Pen-Strep) media containing Dynabeads® Human T-Activator CD3/CD28 (1x105 beads/well) and 10 U/ml recombinant IL-2. Tcon cells (1x105) were also cultured alone as control. 100µl per well was added to round-bottom 96 well plates for each condition. The plates were incubated at 37°C with 5% CO2 for 72 hours.
Post-culture, cells were washed twice and LIVE/DEAD Near-IR Fixable dye was added. Cells were then incubated with Human Fc block (BD Biosciences) prior to extracellular labeling with anti-CD4-PE (A161A1, BioLegend). Cells were washed and fixed with Foxp3 Fixation/Permeabilization Solution (Thermo Fisher Scientific). The cells were then washed in Permeabilization Solution (Thermo Fisher Scientific) and intracellular labeling performed with anti-FOXP3-AF488 antibody (259D, BioLegend). Cells were washed, acquired on the CytoFLEX Flow Cytometer (Beckman Coulter) and analyzed in FlowJo v10.4.
Post-culture, cells were washed twice and LIVE/DEAD Near-IR Fixable dye was added. Cells were then incubated with Human Fc block (BD Biosciences) prior to extracellular labeling with anti-CD4-PE (A161A1, BioLegend). Cells were washed and fixed with Foxp3 Fixation/Permeabilization Solution (Thermo Fisher Scientific). The cells were then washed in Permeabilization Solution (Thermo Fisher Scientific) and intracellular labeling performed with anti-FOXP3-AF488 antibody (259D, BioLegend). Cells were washed, acquired on the CytoFLEX Flow Cytometer (Beckman Coulter) and analyzed in FlowJo v10.4.
10
Intracellular Cytokine Analysis of TILs
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For the intracellular cytokine analysis of TILs, cells from dissociated tumors in the density range of 1×105 to 1×106 cells were cultured in a 96-well U-bottom plate containing 10 µg/mL of pembrolizumab (Selleckchem, Houston, Texas, USA), 10 µg/mL of ipilimumab (Selleckchem), soluble 50 ng/mL of anti-CD3 (BD Biosciences, San Jose, California, USA), and 50 ng/mL of anti-CD28 (BD Biosciences) for 24 hours at 37°C, followed by the addition of brefeldin A (BD Biosciences) and monensin (BD Biosciences) per the manufacturer’s protocol. To measure interferon (IFN)-γ and tumor necrosis factor (TNF) secretion levels by CD8+ TILs according to PD-1 expression on anti-CD3 and anti-CD28 stimulation, the pre-incubation step without brefeldin A and monensin for 24 hours was omitted. After 12 hours, the cells were harvested and stained. Intracellular cytokines were stained after surface staining, fixation, and permeabilization with Foxp3 fixation/permeabilization solution (Thermo Fisher Scientific).
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