Phospho-specific flow cytometry was performed as previously described [40 (link)]. Briefly, after 15 min of treatment, cells were fixed with pre-warmed Fix Buffer I (Becton Dickinson) at 37 °C for 10 min and permeabilized with ice-cold 50% methanol for 30 min on ice. Permeabilized cells were washed with PBS containing 0.1% bovine serum albumin and stained with fluorochrome-conjugated antibodies in the dark at room temperature for 40 min. The complete list of antibodies used, and the staining panels are reported in Supplementary Table S3 and Supplementary Table S4 , respectively. Isotype controls were used to set background signals. Three independent experiments were performed for each cell line and for HD T cells. Approximately 10,000 gated events were acquired for each sample on a FACSCanto cytometer (Becton Dickinson).
Fix buffer 1
Manufactured by BD
Sourced in United States
Fix Buffer I is a laboratory solution used to preserve and stabilize biological samples. It is designed to maintain the structural integrity and chemical properties of proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Perm buffer 3, by BD (39 mentions)
Lsrii flow cytometer, by BD (10 mentions)
Facscalibur, by BD (5 mentions)
Perm buffer, by BD (5 mentions)
Phosflow perm buffer 3, by BD (3 mentions)
Facsaria, by BD (3 mentions)
Gallios flow cytometer, by Beckman Coulter (3 mentions)
Bw264 56, by Miltenyi Biotec (3 mentions)
Polyclonal goat anti mouse ig, by BD (3 mentions)
Bw135 80, by Miltenyi Biotec (3 mentions)
Lsrfortessa, by BD (3 mentions)
Normal mouse serum, by Thermo Fisher Scientific (3 mentions)
Clone 3g8, by BD (3 mentions)
Perm wash buffer, by BD (3 mentions)
Flojo, by Tree Star (2 mentions)
Foxp3 staining buffer, by Thermo Fisher Scientific (2 mentions)
Live dead fixable blue dye, by Thermo Fisher Scientific (2 mentions)
Perm 3 buffer 1, by BD (2 mentions)
Cellquest pro version 4, by BD (2 mentions)
Anti stat1 py701 antibody, by BD (2 mentions)
63 protocols using fix buffer 1
1
Phospho-specific Flow Cytometry Protocol
2
Profiling PD-L1 and pSTAT1 in SV40-Fibroblasts
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SV40-fibroblasts were incubated with the PE-Dazzle-594-PD-L1 (CD274) antibody (clone 29E.2A3, #329732, BioLegend), or the corresponding isotype (#400358, BioLegend). For pSTAT1 staining, the cells were starved overnight in DMEM-1% FBS. For intracellular staining, 106 cells were washed with PBS-2% FBS- 2 mM EDTA buffer, fixed by incubation for 10 minutes at 37°C with Fix Buffer I (#557870, Beckton Dickinson,) and permeabilized by incubation for 20 minutes at 4°C with Phosflow Perm Buffer III (#558050, Beckton Dickinson). Cells were then incubated with PE-coupled STAT1 (clone 1, #558537, Beckton Dickinson) or PE- or AF647-conjugated anti-pSTAT1 antibody (clone 4a, # 612564 or #612597, Beckton Dickinson), the corresponding isotype (#554680 and #565363, respectively, Beckton Dickinson), or unconjugated IRF1 (clone D5E4, #8478, Cell Signaling) or the corresponding isotype, for detection with PE-conjugated goat anti-rabbit antibody (#A10542, Thermo Fisher Scientific). All non-fibroblastic cells were also stained with the Aqua Dead Cell Stain kit (#L34957, Thermo Fisher Scientific). Cells were acquired on a Beckman Coulter Gallios flow cytometer and analyzed with FlowJo Software.
3
Quantifying p-STAT5 in Immune Cells
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To analyze p-STAT5, 100,000 cells were first stained with a viability dye (Fixable Viability Dye eFluor 450, Affymetrix, Thermo Fisher Scientific) for 10 minutes at 4°C. Cells were then fixed with 100 μL of prewarmed (37°C) Fix Buffer I (Becton Dickinson) for 10 minutes at 37°C. Cells were washed once with 1 mL of PBS + 3% FBS. Cells were then permeabilized while vortexing with 100 μL of Perm Buffer III (Becton Dickinson) and incubated for 30 minutes on ice. Cells were washed once with 1 mL of PBS + 3% FBS and stained with 2 μL of p-STAT5–Alexa Fluor 488 (clone 47/Stat5 [pY694]; Becton Dickinson, catalog 612598) in 100 μL of PBS + 3% FBS for 1 hour at room temperature in the dark. Finally, cells were washed once with 1 mL of PBS + 3% FBS, and p-STAT5 was measured by flow cytometric analysis in a FACSCanto II flow cytometer using the FACSDiva software (Becton Dickinson) and analyzed using FlowJo (Tree Star Inc).
4
Targeted Inhibition of IFN Signaling in B Cells
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Example 4
The type I IFN antagonist IFNα2-R120E was fused to the anti-human CD20 nanobody 2HCD25 through a linker sequence made with 20 repeats of GGS motif. The fusion protein was produced in E. coli and purified by Immobilized Metal Affinity chromatography (IMAC).
Human peripheral blood mononuclear cells (PBMCs) are expected to contain ≈4% of B-cells which can be characterized by the cell surface expression of CD19. The large majority of circulating B-cells are also positive for the expression of CD20.
PBMCs were isolated over ficoll gradient (histopaque-1077, Sigma-Aldrich) from blood samples of healthy donors. Cells were left untreated or were incubated for 15 minutes with 50 pM of human IFNα2 in the absence or presence of 10 μg/ml of the 2HCD25 nanobody-IFNα2-R120E fusion protein.
Cells were then fixed (BD Fix Buffer I), permeabilized (BD Perm Buffer III) and labelled with PE-labelled anti pSTAT1 (BD #612564) and APC-labelled anti human CD19 (BD #555415). FACS data were acquired using a BD FACS Canto and analyzed using Diva (BD Biosciences) software for the fluorescence associated with pSTAT1 in CD19 positive and negative cell populations.
5
Characterization of Cultured TIL Phenotypes
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The cultured TIL phenotypes after culture were characterized using flow cytometry with anti-CD3 (Cat#: 555339, 1.5 μL/106 cells), anti-CD4 (Cat#: 557871, 2 μL/106 cells), anti-CD8 (Cat#: 563823, 2 μL/106 cells), anti-CD56 (Cat#: 56275, 3 μL/106 cells), and anti-PD1 (Cat#: 561272, 5 μL/106 cells) for 30 minutes on ice in the dark [35 (link), 37 (link)]. Thereafter, the cells were washed once with PBS and resuspended in 400 μL PBS. 7AAD was used to distinguish live cells and dead cells, and the cells were run on a BD Fortessa (BD Biosciences). Fluorescence minus one (FMO) was used as the negative control. Moreover, FlowJo software was used to analyze the data generated by flow cytometry. FoxP3 staining was conducted using an intracellular staining protocol from BD Biosciences as follows: anti-CD3 and anti-CD4 were stained for 30 minutes on ice in the dark; TILs were washed, fixed, and permeabilized following protocols for BD Fix Buffer I (Cat#: 557870, BD Biosciences, USA) and Perm Buffer III (Cat#: 558050, BD Biosciences, USA). The cells were washed thrice with Perm Buffer III and incubated with anti-FoxP3 (Cat#: 560460, 5 μL/106 cells) for 30 minutes on ice in the dark. The cells were run on a BD Fortessa (BD Biosciences). Fluorescence minus one (FMO) was used as the negative control. FlowJo software was used to analyze the data generated by flow cytometry.
6
Mutagenesis and FACS Sorting of HAP1 Cells
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HAP1 cells were mutagenized to generate a library of cells that carry insertional mutations. The resulting cells were expanded to approximately 1.5 × 109 cells and subsequently dissociated using trypsin-EDTA (Life Technologies, Cat# 15090046), washed with PBS, and stained with anti-CD58-APC antibody (Thermo fisher scientific, Cat# 17-0578-42). Subsequently, cells were washed three times with PBS containing 1% FCS, passed through a 40 μm strainer (BD Falcon™, Cat# 352340), and subsequently fixed using BD fix buffer I (BD biosciences, Cat# 557870) for 10 min at 37°C, followed by a wash with PBS containing 1% FCS. The staining of the cells was finished with a final wash in PBS containing 10% FCS. The cell sorting and downstream processing were conducted as described in Brockmann et al.60 (link)
7
Phosphorylation Analysis of Tregs
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Flow cytometry was performed with directly conjugated Abs as previously described (6 (link)). A fixable Blue LIVE/DEAD dye (Life Technologies) was used to exclude dead cells from analysis. FOXP3 and CTLA-4 (total) staining was performed using the eBioscience FOXP3 staining buffers. For staining of phosphorylated proteins, BD Fix Buffer I and BD Phosflow perm buffer III were used according to manufacturer’s instructions. In brief, sorted Tregs (at 5 × 105/ml in 100 μl RPMI 1640) were incubated for 15 min with either SF or 10 ng/ml rhIL-6 before addition of 100 μl BD Fix Buffer I and incubated for 10 min at 37°C. Cells were washed extensively in PBS, then permeabilized by addition of 1 ml ice-cold Perm Buffer III and incubation for 30 min on ice. Cells were washed three times and then stained for PE–anti–p-STAT3 or PE–anti–p-STAT5. Data were collected on the LSR II, FACS Aria, or FACSCalibur flow cytometers (all BD Biosciences). Flow cytometry data were analyzed using FloJo (Tree Star) software.
8
Flow Cytometry Analysis of Immune Cells
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Single cell suspensions derived from pLN, mLN, and spleen of host mice were stained with the indicated antibodies in PBS containing 0.5% FBS and 0.2% sodium azide. The following antibodies conjugated to various fluorophores were used for analysis: CD4 (RM4-5), CD8 (53-6.7), CD31 (MEC13.3), CD35 (7E9), gp38 (8.1.1), CD44 (IM7), Vα2 (B20.1), CD45.1 (A20), CD127 (A7R34) and CD90.1 (OX-7). Donor T cells were identified using anti-CD45.1 and CD90.1 antibodies. Anti-Vα2 was included to identify TCR transgenic donor T cells. Non-specific binding was blocked using 2.4G2 and propidium iodide was added to eliminate dead cells from the analysis. Intracellular staining for phosphorylated STAT5 was performed by fixing isolated cells with BD Fix Buffer I for 10 minutes at 37 °C followed by permeabilization in 90% methanol for 1 hour at 4 °C. After washing, cells were labeled overnight at 4 °C with a pY694-STAT5-PE antibody (BD biosciences). Isolation and staining of stromal cells was performed as previously described11 (link). Data from stained cells was collected using a BD LSR-II flow cytometer.
9
Phospho-flow Cytometry of Treg Cells
10
Multiparametric Flow Cytometry Analysis
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