Heparinized whole blood was collected before 10 a.m. during the mid-luteal period of the menstrual cycle. Whole blood (100 μL) was incubated for 15 min with antibodies against the cell surface markers CD3 (5 μL) and CD56 (5 μL), then the blood was lysed using 1x FACS lysis solution (349202; BD Pharmingen). Cells were permeabilized with 1x Permeabilizing Solution 2 (340973; BD Pharmingen) for 10 min, then incubated for 30 min with antibodies against perforin, granzyme B and granulysin (10 μL each). Cells were analyzed on BD FACSCanto II flow cytometer using BD FACSDiva software.
Permeabilizing solution 2
Manufactured by BD
Sourced in United States
Permeabilizing Solution 2 is a laboratory product designed to temporarily increase the permeability of cell membranes. It facilitates the introduction of molecules, such as antibodies or other reagents, into the interior of cells for various analytical and experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lysing solution, by BD (7 mentions)
Lsrfortessa, by BD (5 mentions)
Facscanto 2 flow cytometer, by BD (4 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (4 mentions)
Facs lysing solution, by BD (3 mentions)
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Facs lysis solution, by BD (1 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Vectashield containing 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Anti cd8 apc, by BioLegend (1 mentions)
31 protocols using permeabilizing solution 2
1
Lymphocyte Phenotyping by Flow Cytometry
2
Multi-Dimensional Cell Immunophenotyping
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Cell surface was stained at room temperature for 30 min. In case of subsequent intracellular cytokine staining, cells were fixed for 10 min with 2% paraformaldehyde and permeabilized using 1x Permeabilizing Solution 2 (BD Biosciences) at room temperature for 10 min. Cytokine staining was performed at room temperature for 30 min. Transcription factors were stained using eBioscienceTM FoxP3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to manufacturer’s instructions. A full list of antibodies used can be found in Supplementary Table 2 . Stained samples were acquired on BD LSR FortessaTM equipped with 355-, 405-, 488-, 561-, and 639-nm lasers. Analysis was performed using FlowJo v. 9.9 (TreeStar).
3
Visualizing HER3 and EGFR/HER2 in Breast Cancer Cells
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Human HER3+ breast cancer cells (SKBR3, BT474M1 and MDA-MB-468) were incubated with 1:100 HER3-VIA or GFP-VIA at 37 °C for 60 min (SKBR3, BT474M1) or 3 h (MDA-MB-468). After washing, fixation with 4% paraformaldehyde (PFA), and application of permeabilizing solution 2 (Becton Dickenson), nonspecific binding was blocked with 2.5% goat serum at 37 °C for 30 min. Cells were incubated with 1:100 Red™-conjugated anti-mouse IgG (H + L) (Jackson ImmunoResearch Laboratories Inc. West Grove, PA, USA) in a dark chamber for 1 h at room temperature and washed with PBS. For the detection of EGFR, MDA-MB-468 cells were incubated with HER3-VIA for 3 h, then fixed and permeabilized. Cells were then labeled with cetuximab (40 μg/mL) for 10 min, followed by staining with Cy2-conjugated anti-human IgG antibody (ab97169, Abcam, Cambridge, MA, USA). For the detection of HER2, SKBR3 cells were labeled with trastuzumab (40 μg/mL) for 10 min, washed with medium, and then incubated with HER3-VIA for 3 h. Then, cells were fixed and permeabilized, and stained with Cy2-conjugated anti-human IgG antibody. Slides were mounted in VectaShield containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) and images were acquired using a Zeiss Axio Observer wide-field fluorescence microscope (Carl Zeiss, München-Hallbergmoos, Germany).
4
Multiparametric T Cell Phenotyping
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The cells were subsequently incubated with EDTA (final concentration 1.4 mM) at RT. Afterwards the cells were permeabilized (Becton Dickinson Permeabilizing solution 2) and stained with anti-CD3–allophycocyanin(APC)/Cy7, anti-CD4-peridinin chlorophyll(PerCp), anti-CD8-APC, anti-CD69-R-phycoerythin(PE), and anti-IFN-γ-fluorescein isothiocyanate(FITC) (Biolegend, San Diego, USA). Finally the cells were fixed in 1% formaldehyde and analyzed by flow cytometry on LSRII (BD Biosciences).
5
Peptide-Stimulation Assay for T-Cell Response
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In vitro cultured PBMC's were harvested, washed, and aliquoted at 2–4 × 105 cells/well in a round bottom 96 well-plate. The in vitro cultured PBMC's were stimulated for 4 h at 37°C, 5% CO2 with, for the matrix analysis, each of the 15 column and 15 row mixes (1 μM/peptide), and for the epitope identification with a single peptide (0.8 μM). After 1 h of stimulation 1 μg/mL Brefeldin A (BD Biosciences) was added. The cells were subsequently permeabilized (Becton Dickinson Permeabilizing solution 2) and stained with anti-CD3-Pacific Blue, anti-CD4-PerCp, anti-CD8-APC, anti-CD69-PE, and anti-IFNγ-FITC, according to the “FastImmune” protocol (Becton Dickinson). The cells were subsequently analyzed by flow cytometry using a LSRII (BD Biosciences). The gating strategy is illustrated in Supplementary Figure S4 . Staining of more than 0.8% of CD8+ or CD4+ T cells was considered positive.
6
T cell activation and IFN-gamma analysis
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In vitro cultured PBMC's were harvested, washed, and aliquoted at 2-4 x 10 5 cells/well. The cells were incubated with relevant peptide matrix column and row mixes (1uM/peptide) or single peptide (0.8 uM) for 4 h at 37°C, 5% CO 2 . Brefeldin A was added for the last 3 h of incubation. The cells were subsequently permeabilized (Becton Dickinson Permeabilizing solution 2) and stained with anti-CD3, -CD4, -CD8, -CD69, and -IFNγ, according to the "FastImmune" protocol (Becton Dickinson). The cells were subsequently analyzed by flow cytometry using a LSRII (BD Biosciences). Staining of more than 0.8% of CD8 + or CD4 + T cells was considered positive.
7
Immunophenotyping of Regulatory T Cells
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Regulatory T cell subsets were defined as CD4+CD25highFoxP3+ and CD4+FoxP3+CTLA-4+ cells. Directly after isolation, PBMCs were first aliquoted into tubes without PMA + ion stimulation, and then surface-stained with PerCP anti-human CD4 and FITC anti-human CD25 or FITC anti-human CTLA-4 mAbs. After fixation with 2 % PFA and permeabilization with BD Permeabilizing Solution 2 (Becton Dickinson), the cells were incubated with PE anti-human FoxP3 mAb.
After staining, the cells were washed and immediately analyzed with a FACScan cytometer equipped with Cell Quest software (BD Bioscience Pharmingen). In each case, staining was compared with that of the appropriately labeled isotype control. Lymphocytes were gated on the basis of forward- and side-scatter properties, and at least 30,000 CD4+ T cells were analyzed.
After staining, the cells were washed and immediately analyzed with a FACScan cytometer equipped with Cell Quest software (BD Bioscience Pharmingen). In each case, staining was compared with that of the appropriately labeled isotype control. Lymphocytes were gated on the basis of forward- and side-scatter properties, and at least 30,000 CD4+ T cells were analyzed.
8
Th1 and Th17 Cell Identification
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Before incubation of the cells with phorbol 12-myristate 23-acetate (PMA), CD4+ T cells were purified by negative selection with CD4+ T cell isolation kit by magnetic cell sorting (Miltenyi Biotec) to avoid PMA-mediated internalization and degradation of the CD4 molecule, which would affect the identification of Th1 (CD4+IL-17-IFN-gamma+) and Th17 (CD4+IFN-gamma-IL-17+) cells [28 (link)]. Then, the cells were stimulated with 25 ng/ml PMA and 1 μg/ml of ionomycin (Ion) (Sigma-Aldrich) in the presence of 10 μg/ml of brefeldin A (BFA, protein transport inhibitor) and cultured for 4 h at 37 °C in a humidified 5 % CO2 incubator followed by the cells’ fixation and permeabilization with BD Permeabilizing Solution 2 (Becton Dickinson) according to the manufacturer’s instruction. Next, the cells were stained with phycoerythrin (PE)-labeled anti-human IL-17 (eBioscience) and fluorescein isothiocyanate (FITC)-labeled anti-human IFN-γ (Becton Dickinson) monoclonal antibodies (mAbs).
9
Multiparametric Flow Cytometry Analysis
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Single cell suspensions were stained with different combinations of fluorochrome-conjugated antibodies (Table S1 in Supplementary Material), and dead cells were excluded using Fixable Dead Cell Stain Kit (Thermo Fisher) or Fixable Viability Kit (BioLegend). For intracellular staining of IFN-γ and TNF, cells were surface stained and fixated with 2% PFA (EMS Sciences), followed by treatment with Permeabilizing Solution 2 (BD Biosciences). Data were acquired on an LSR Fortessa (BD Biosciences) and analyzed with FlowJo vX (FlowJo LLC) software. ARIA or ARIA II instruments (both BD Biosciences) were used for cell sorting experiments.
10
Flow Cytometric Analysis of MMP-9 and TIMP-1
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Cells were stained with the following monoclonal antibodies; CD3- APC/H7 clone SK7 (BD Biosciences, San José, CA, US) and CD14-PeCy7 clone M5E2 (Nordic Biosite, Täby, Sweden). Whole blood and antibodies were incubated for 15 minutes at RT, thereafter erythrocytes were lysed with FACS Lysing Solution (BD Biosciences) for 15 minutes at room temperature (RT). The cells were permeabilized for 30 min at RT with Permeabilizing Solution 2 (BD Biosciences) and washed with Permeabilization buffer (Ebioscience, San Diego, CA, USA). Unspecific binding was blocked with 10% FCS and thereafter, cells were stained with antibodies against MMP-9 and TIMP-1, conjugated with FITC and PE respectively, (RnD Systems) for 30 min in 4°C. After washing in Permeabilization buffer followed by resuspension in PBS with 0.5% FCS, cells were analyzed using Beckman Coulter Gallios (Beckman Coulter, Miami Lakes, Florida, US). The obtained data was analyzed and visualized using the Kaluza 1.2 software (Beckman Coulter). When this analysis was performed, antibodies against TIMP-2 were not commercially available.
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