Ex vivo cultured erythroblasts were collected at the indicated time points as described above. Before fixation, cells were stained for cell surface marker TER119‐APC in PBS/2%FBS for 15 min at RT. After washing, cells were fixed in 4% FA/PBS as described above and stored in PBS/2%FBS overnight at 4°C. Fixed cells were permeabilized by adding 200 µl 1× eBioscience Permeabilization Buffer (00‐8333‐56, Thermo Fisher Scientific) and pelleted by centrifugation for 5 min at 300 g. Cells were stained for TER119‐APC, Hoechst, and pHH3‐Alexa Fluor 488 in 1× eBioscience Permeabilization Buffer for 20 min at RT. After washing, cells were resuspended in PBS/2%FBS and acquired using ImageStream X as described above. Per sample, 30,000–40,000 events per cell gate were collected. Analysis of mitotic stages was performed as described by Filby et al (2011 (link)).
Ebioscience permeabilization buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EBioscience™ Permeabilization Buffer is a reagent used to permeabilize cells for intracellular staining and analysis. It is designed to allow antibodies or other probes to access the intracellular compartments of cells.
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Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Cd8 fitc, by BD (2 mentions)
Cd3 apc h7, by BD (2 mentions)
Cd4 percp, by BD (2 mentions)
Ebioscience fixation permeabilization buffers, by Thermo Fisher Scientific (2 mentions)
Fvs510, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Ebioscience ic fixation buffer, by Thermo Fisher Scientific (2 mentions)
Anti rabbit af488, by Thermo Fisher Scientific (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Antibody rabbit anti ttr, by Abcam (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Facscan flow cytometer, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Ebioscience intracellular fixation buffer, by Thermo Fisher Scientific (2 mentions)
Cytofix, by BD (2 mentions)
Anti klrg1 pecy7 clone 2f1, by Thermo Fisher Scientific (2 mentions)
Anti ctla 4 pe clone uc10 4b9, by Thermo Fisher Scientific (2 mentions)
Anti cx3cr1 bv711 clone sa011f11, by BioLegend (2 mentions)
Fortessa flow cytometer, by BD (2 mentions)
25 protocols using ebioscience permeabilization buffer
1
Erythroblast Mitotic Phase Profiling
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Ex vivo cultured erythroblasts were collected at the indicated time points as described above. Before fixation, cells were stained for cell surface marker TER119‐APC in PBS/2%FBS for 15 min at RT. After washing, cells were fixed in 4% FA/PBS as described above and stored in PBS/2%FBS overnight at 4°C. Fixed cells were permeabilized by adding 200 µl 1× eBioscience Permeabilization Buffer (00‐8333‐56, Thermo Fisher Scientific) and pelleted by centrifugation for 5 min at 300 g. Cells were stained for TER119‐APC, Hoechst, and pHH3‐Alexa Fluor 488 in 1× eBioscience Permeabilization Buffer for 20 min at RT. After washing, cells were resuspended in PBS/2%FBS and acquired using ImageStream X as described above. Per sample, 30,000–40,000 events per cell gate were collected. Analysis of mitotic stages was performed as described by Filby et al (2011 (link)).
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Ex vivo cultured erythroblasts were collected at the indicated time points as described above. Before fixation, cells were stained for cell surface marker TER119‐APC in PBS/2%FBS for 15 min at RT. After washing, cells were fixed in 4% FA/PBS as described above and stored in PBS/2%FBS overnight at 4°C. Fixed cells were permeabilized by adding 200 µl 1× eBioscience Permeabilization Buffer (00‐8333‐56, Thermo Fisher Scientific) and pelleted by centrifugation for 5 min at 300 g. Cells were stained for TER119‐APC, Hoechst, and pHH3‐Alexa Fluor 488 in 1× eBioscience Permeabilization Buffer for 20 min at RT. After washing, cells were resuspended in PBS/2%FBS and acquired using ImageStream X as described above. Per sample, 30,000–40,000 events per cell gate were collected. Analysis of mitotic stages was performed as described by Filby et al (2011 (link)).
2
Analyzing T Cell Phenotypes by ICS
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To evaluate the phenotypes of T cells that responded to stimulation in IFN-γ ELISPOT assays, we conducted intracellular cytokine staining (ICS). The in vitro stimulation was similar to that described above in the IFN-γ ELLISPOT section. Briefly, after 12-14 days in vitro culture, PBMCs were restimulated with peptide. After 1 h, Brefeldin A was added to inhibit protein transport. After 4 additional hours of incubation, the cells were stained with T-cell surface markers CD4-PerCP (cat. 345770), CD8- FITC (cat. 345772), CD3-APC-H7 (cat. 560275) and a dead cell marker FVS510 (564406) (all from BD Biosciences). Samples were then fixed and permeabilized using eBioscience™ Fixation/Permeabilization buffers (eBioscience, cat. 00-5123-43, 00-5223-56) and stained with IFNg-APC (cat.341117, BD Biosciences), TNFa-BV421 (cat.562783, BD Biosciences) in eBioscience permeabilization buffer (eBioscience, cat. 00-8333-56) and analyzed on FACSCanto™ II (BD Biosciences) using BD FACSDiva software version 8.0.2 as described previously (32 (link)).
3
Peptide-Specific T Cell Response Phenotyping
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ARG1 peptide-specific T cell response phenotype was tested by intracellular cytokine staining (ICS) of PBMCs stimulated with the individual 20-mer peptides. Similar to ELISPOT assay, prior to being used in the ICS assay, PBMCs were stimulated in vitro with the individual 20-mer peptides and low dose IL-2 (120U/ml) for 14 days. After 14-day culture, PBMCs were stimulated with 5 μM of the appropriate ARG1 peptide or for five hours in a 96-well plate. One hour after adding the peptide, the protein transport inhibitor BD GolgiPlug™ (BD Biosciences) was added. Non-stimulated PBMCs were used as a control to determine the background cytokine levels. Following the 5-hour incubation, the PBMCs were stained using the following antibodies: CD3-APC-H7 (BD Biosciences), CD4-PerCP (BD Biosciences), CD8- FITC (BD Biosciences). The dead cells were stained with FVS510 (BD Biosciences). Stained samples were then fixed and permeabilized overnight using eBioscience™ Fixation/Permeabilization buffers (eBioscience) as stated by the instructions of the manufacture. The following day cells were stained intracellularly using the eBioscience permeabilization buffer (eBioscience) with TNFα-BV421 (BD Biosciences) and IFNγ-APC (BD Biosciences). The samples were analyzed using the FACSCanto™ II (BD Biosciences) with BD FACSDiva software (v. 8.0.2).
4
Identifying Murine Neutrophils by Flow Cytometry
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Mice were anesthetized by inhalation with isoflurane, and blood from the hearts of mice was placed in heparin-coated tubes and treated with RBC lysis buffer. The following monoclonal antibodies were used for flow cytometric analysis at a dilution of 1/400: anti-CD45, anti-CD11b, anti-Ly6G, anti-Ly6C, anti-Ly6G/Ly6C (Gr-1) antibodies. For intracellular staining, after staining with antibodies against cell surface proteins, cells were washed three times with MACS buffer, fixed with eBioscience™ IC Fixation Buffer (Thermo Fisher Scientific), permeabilized with eBioscience™ Permeabilization Buffer, and then stained with an antibody against an intracellular marker. Cells not stained with FVD780 were categorized as live cells. Data were acquired with LSRFortessa (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo. The gating strategies of neutrophils (CD45+CD11b+Ly-6Gintra+) are shown in Supplementary Figure S4 .
5
Multicolor Flow Cytometry Analysis
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Cells were resuspended in FACS buffer, and stained as previously described [39 (link), 40 (link)]. In short, cells were incubated with an anti-mouse CD16/32 unconjugated antibody (clone 93, BioLegend) and stained with fixable viability dye Zombie NIR (BioLegend), for 20 min at 4 °C. Subsequently, cells were stained (30 min, 4 °C) using fluorochrome-conjugated antibodies. CD11b (PerCP-Cy5.5, clone M1/70) purchased from eBioscience, CD45 (BV510, clone 30-F11), MHCII (BV711, clone M5/114.15.2), VE-cadherin (BV421, clone BV13), and gp38 (AF488, clone PMab-1) purchased from BioLegend. CD31 (APC, clone MEC13.3) purchased from BD Biosciences. Cells were washed (400 × g, 5 min 4 °C) with FACS buffer, fixed in 4% PFA for 15 min at 4 °C and re-suspended in FACS buffer. For CP, cells were additionally permeabilized with eBioscience™ Permeabilization buffer (Invitrogen), incubated with antibody rabbit-anti-TTR (Abcam) for 40 min, and stained with secondary antibody anti-rabbit AF488 (ThermoFisher). Cells were acquired using Attune NxT Flow Cytometer (ThermoFischer). Data were analyzed using FlowJo software (version 10.5.3, FlowJo LLC, OR, USA).
6
Characterizing Lymphocyte Populations in TIL
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To characterize lymphocyte populations within autologous TIL, cells were harvested (up to 1 × 106 cells/staining depending on available number of cells), washed 2× with PBS (200 rpm, 5 min at 4 °C), resuspended in staining buffer (PBS plus 10% FBS) and plated in a 96-well V-bottom plate (100 μL/well) (Corning). To verify >90% cell viability, cells were counted with a Nucleocounter (Chemotec) before plating. For each panel staining, an unstained control and, if necessary, a FMO control were prepared. For extracellular staining, cells were incubated with antibodies (see SI Materials ) for 30 min at 4 °C in the dark. For subsequent intracellular staining, cells were washed 2–3 times (200 rpm, 5 min at 4 °C) in eBioscience™ Permeabilization buffer (250 µL/well) (Invitrogen) and resuspended in eBioscience™ Fixation/Permeabilization solution (Invitrogen) for 20 min at 4 °C. After 2–3 washing steps (200 rpm, 5 min at 4 °C), cells were incubated with antibodies (30 min, 4 °C in dark) (see SI Materials ). After the staining process, cells were washed 2–3 times and analyzed with a BD FACS Melody machine (BD Biosciences, Franklin Lakes, NJ, USA).
7
TRAIL-R Membrane Expression and Apoptosis
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For detection of TRAIL-Rs membrane expression, cells were incubated with primary antibodies for 45 min on ice (or at 37 °C where indicated). For detection of total TRAIL-R2 expression, cells were permeabilized with eBioscience Permeabilization Buffer (Invitrogen) before staining. Antibodies (Supplementary Table 1 ) were diluted as recommended. Fluorescence index was calculated as ratio between mean fluorescence intensity values of TRAIL-Rs and negative controls (IgG isotype PE-conjugated or ALEXA488 in direct or indirect immunostaining, respectively).
Quantification of apoptosis was carried out by staining cells with FITC-conjugated Annexin V (Abcam) and To-PRO-III cyanine dye (Invitrogen). Mitochondrial membrane potential (ψm) was determined by staining cells with 5 μg/ml JC-1 dye (Invitrogen) for 10 min at 37 °C.
Fluorescence labeling was measured acquiring samples by the LSRFortessa system (Becton-Dickinson), data were analyzed and processed with the FlowJo software (Tree Star Inc).
Quantification of apoptosis was carried out by staining cells with FITC-conjugated Annexin V (Abcam) and To-PRO-III cyanine dye (Invitrogen). Mitochondrial membrane potential (ψm) was determined by staining cells with 5 μg/ml JC-1 dye (Invitrogen) for 10 min at 37 °C.
Fluorescence labeling was measured acquiring samples by the LSRFortessa system (Becton-Dickinson), data were analyzed and processed with the FlowJo software (Tree Star Inc).
8
Zombie Aqua Fixation and Antibody Staining
9
Comprehensive Immune Cell Phenotyping
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Part of the cells were first permeabilized with 0.5% saponin (Sigma-Aldrich) or eBioscience permeabilization buffer (Invitrogen). After washing, cells were next stained with antibodies directed against CD45, CD45RA, CD45RB, CD45RC, CD45.2, CD64 , Siglec-F , CD11b, Ly6C, F4/80 and CD11c in permeabilization buffer. Cells were measured on a FACSCanto flow cytometer or LSR-II (both BD Biosciences), and gates were set according to Fluorescence Minus One (FMO) controls.
10
Multiparametric Flow Cytometry Analysis
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Cells were resuspended in FACS buffer, and stained as previously described (39, 40) .
All antibodies were purchased from BioLegend unless otherwise stated. In short, cells were incubated with an anti-mouse CD16/32 antibody (clone 93, BioLegend, 101302) and stained with fixable viability dye ZombieNIR, for 20 min at 4°C. Subsequently, cells were stained (30 min, 4 °C) using fluorochrome-conjugated antibodies CD11b (PerCP-Cy5.5) eBioscience, CD45 (BV510), MHCII (BV711), and CD31 (APC) BD Biosciences. Cells were washed (400 g, 5 min 4°C) with FACS buffer, fixed in 4% PFA for 15 min at 4 °C and re-suspended in FACS buffer. For CP, cells were additionally permeabilized with eBioscience™ Permeabilization Buffer (Invitrogen), incubated with antibody rabbit-anti-TTR (Abcam) for 40 min, and stained with secondary antibody anti-rabbit AF488 (ThermoFisher). Cells were acquired using Attune NxT Flow Cytometer (Thermo Fischer). Data was analysed using FlowJo software (version 10.5.3, FlowJo LLC, OR, USA).
All antibodies were purchased from BioLegend unless otherwise stated. In short, cells were incubated with an anti-mouse CD16/32 antibody (clone 93, BioLegend, 101302) and stained with fixable viability dye ZombieNIR, for 20 min at 4°C. Subsequently, cells were stained (30 min, 4 °C) using fluorochrome-conjugated antibodies CD11b (PerCP-Cy5.5) eBioscience, CD45 (BV510), MHCII (BV711), and CD31 (APC) BD Biosciences. Cells were washed (400 g, 5 min 4°C) with FACS buffer, fixed in 4% PFA for 15 min at 4 °C and re-suspended in FACS buffer. For CP, cells were additionally permeabilized with eBioscience™ Permeabilization Buffer (Invitrogen), incubated with antibody rabbit-anti-TTR (Abcam) for 40 min, and stained with secondary antibody anti-rabbit AF488 (ThermoFisher). Cells were acquired using Attune NxT Flow Cytometer (Thermo Fischer). Data was analysed using FlowJo software (version 10.5.3, FlowJo LLC, OR, USA).
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