For cytometric analysis, the cells were washed with PBS (2000 rpm, 10 min). To detect internal antigens, the cells were permeabilized using formaldehyde/saponin-based permeabilization IntraPrep™ Kit (Beckman-Coulter). The macrophage lineage J774A.1 was externally labeled with anti-CD86-APC and anti-CD14-FITC. The HT-29 line was externally and internally labeled with anti-TLR-4-PE and anti-TLR2-FITC. Mouse IgG conjugated to FITC, PE, or APC was used as isotype control. PBMCs were externally labeled with anti-CD4-FITC, anti-CD25-PE, and intracellular anti-Foxp3-PE staining. Analyses were made in FC500 Beckman-Coulter cytometer. Data were processed using the Kaluza® flow analysis software.
Intraprep kit
Manufactured by Beckman Coulter
Sourced in United States
The IntraPrep kit is a product manufactured by Beckman Coulter. It is a laboratory equipment designed for the preparation of intracellular antigens.
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Lab products found in correlation
Brefeldin a, by BioLegend (3 mentions)
Golgiplug, by BD (2 mentions)
Anti pan cytokeratin pan ck pe, by Santa Cruz Biotechnology (2 mentions)
Facs navio fc, by Beckman Coulter (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Pe conjugated anti ifn γ, by Beckman Coulter (1 mentions)
Facscanto 10c system, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
Facscalibur system, by BD (1 mentions)
Anti cd3 fitc, by BioLegend (1 mentions)
Anti cd8 apc cy7, by BioLegend (1 mentions)
Anti cd4 percp, by BioLegend (1 mentions)
Anti tnf α apc, by BioLegend (1 mentions)
Tlr7 antibody, by Novus Biologicals (1 mentions)
Cd34 pe antibody, by Beckman Coulter (1 mentions)
Alexa fluor 647, by Santa Cruz Biotechnology (1 mentions)
Cd44 fitc, by BioLegend (1 mentions)
21 protocols using intraprep kit
1
Multiparametric Flow Cytometry Analysis
2
MAGE-A4 Peptide Stimulation of CD8+ T Cells
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Target cells (T2A24) were pulsed with 4 μg/μL MAGE-A4143–151 peptide for 3 h and then were cocultured with effector cells at an effector/target ratio of 1:1. Two hours later, GolgiPlug (BD Biosciences) was added to the samples at 1 μL/mL and then the cells were further incubated for another 20 h. For staining, the cells were first stained with FITC-conjugated anti-CD8 and then permeabilizated using a IntraPrep kit (Beckman Coulter, Brea, CA, USA) following the manufacturer’s instructions. The cells were further stained intracellularly with PE-conjugated anti-IFN-γ (Beckman Coulter) and analyzed by flow cytometric analysis.
3
Quantifying Histone H2A.X Phosphorylation
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Phosphorylation of the histone H2A family member H2A.X (γH2A.X) at Ser 139 was measured by flow cytometry. Briefly, cells were fixed and permeabilized using an IntraPrep kit (Beckman Coulter). The cells were incubated with Alexa Fluor 488-conjugated anti-phospho-γH2A.X (Ser 139) antibody (Cell Signaling Technology, Danvers, MA, USA) and analyzed by FACSCalibur.
4
Peptide-Stimulated PBMC Cytokine Response Analysis
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After overnight resting, 1x106 isolated PBMCs were stimulated with peptide pools derived from SARS-CoV-2 M, N, S, S1 and S2 proteins (Miltenyi Biotec and JPT) at a final concentration of 1 μg of each peptide per ml peptide pool. Unstimulated PBMCs served as negative controls, and cells stimulated with phorbol 12-myristate 13-acetate (PMA; 10 ng/mL) and ionomycin (500 ng/mL, Sigma Aldrich) served as positive controls. Antigens from epitopes of huCoV strains OC43 and 229E as well as RSV_NP, IAV_MP1 (all JPT) and CMV_pp65 (Miltenyi Biotec) were also analyzed. After 1 h of incubation, 5 μg/mL Brefeldin A was added to each well (BioLegend). Following a total stimulation time of 5 h, cells were harvested and extracellularly stained with anti-CD45 Pacific Blue, anti-CD4 PerCP, anti-CD8 PE-Cy7, anti-CD45RA BV605 and anti-CD62L FITC. Subsequently, cells were fixed and permeabilized using the IntraPrep Kit according to the manufacturer’s instructions (Beckman Coulter). Cells were intracellularly stained with anti-IFN-γ PE and anti-TNF-α APC. Samples were acquired on a FACSCanto 10c system (BD Biosciences), and at least 50,000 events in the CD45+ lymphocyte gate were analyzed in each test.
5
Hypoxia Assessment in Amniotic Cells
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The hypoxic status of AM and AF cells was assessed using Hypoxyprobe-1 Plus Kit (HPI). Pregnant mice (E13.5) were intravenously injected with 120 mg kg−1 of piminidazole (Pimo) 2 hours before euthanasia. Cells obtained from AF and AM were used for FACS analysis or stained. For the FACS analysis, cells were permeabilized using IntraPrep Kit (Beckman Coulter) following the manufacturer’s procedure, while for immunofluorescence onto cytospin AF or AM cells were fixed using 4% PFA and permeabilized with 0.1% triton X-100. Detection of intracellular Pimo adducts was performed labeling with FITC-conjugated mouse anti-Pimo antibody. PBS-injected mice were used as controls to detect baseline levels of anti-Pimo antibody binding.
6
Identification of FcRγ-deficient NK Cells
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Signaling adaptors (FcRγ and CD3ζ) were detected by using a modification of the previously described method [4 (link)]. Instead of using peripheral blood mononuclear cells (PBMCs), whole blood samples (50 µL) were stained for flow cytometry analysis by using phycoerythrin (PE)-conjugated anti-CD3 (clone UCHT1; BD Biosciences, San Jose, CA, USA) and PE-conjugated Cy5-anti-CD56 antibodies (clone B159, BD Biosciences). After surface labeling, the cells were washed, fixed, and permeabilized (IntraPrep kit, Beckman Coulter; Fullerton, CA, USA), according to the manufacturer's instructions. For detection of intracellular FcRγ and CD3ζ expression, fixed and permeabilized cells were stained with fluorescein isothiocyanate (FITC)-anti-FcεRIγ (FcRγ) (Millipore, Temecula, CA, USA) or FITC-anti-CD247 (CD3ζ) (Biolegend, San Diego, CA, USA) antibodies. Samples were acquired with FACSCalibur system (Becton Dickinson, San Jose, CA, USA), and the resulting data were analyzed by using CellQuest software (Becton Dickinson). On the basis of these two signal adaptors' intracellular expression of CD3-/CD56dim NK cells, a distinct subset of human NK cells were identified as g-NK cells; these cells are deficient for FcRγ expression, but express normal levels of CD3ζ. We used 10% as an arbitrary cut-off value to define the presence or absence of g-NK cells for this study.
7
Quantification of CMV-specific T-cells
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After 3 days of incubation in the presence or absence of vitamin C, 1 × 106 cells per sample were stimulated with peptide pool CMV pp65pp for one hour followed by addition of Brefeldin A (BioLegend) and incubation for another 15 h at 37 °C and 5% CO2. Cells were resuspended, washed and extracellularly stained using FITC anti-CD3, APC/Cy7 anti-CD8 and PerCP anti-CD4 (all BioLegend). Subsequently, cells were fixed, permeabilized and intracellularly stained with APC anti-TNF-α (BioLegend) using IntraPrep kit (purchased by Beckman Coulter) following the manufacturer’s instructions. Samples were acquired on a FACS Canto 10c, and gates were set based on the forward scatter versus side scatter properties of lymphocytes. At least 30,000 events were acquired in the CD3+ gate. Data were analyzed using FlowJo_v10.7.1 software.
8
Flow Cytometric Analysis of TLR7 and TLR8
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Cells derived from normal pancreas, chronic pancreatitis and pancreatic cancer tissues were analyzed on a flow cytometer (Beckman Coulter, Krefeld, Germany) with a software package (Coulter, Epics XL-MCL, System II). TLR7 antibody was purchased from Imgenex, TLR8 was provided by ProSci. CD34-PE antibody, FITC-conjugated anti-rabbit secondary antibody and isotype control antibodies were purchased by Beckman Coulter. For intracellular staining we used IntraPrep kit (Beckman Coulter).
9
Flow Cytometry Analysis and Sorting Methodology
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Flow cytometry analysis was performed with the BD™ LSRII cytometer. Cells were detached with TrypLE™ Express Enzyme (Cat# 12604013, Gibco) and suspended in PBS. Cells were stained with Live/Dead fixable dead cell stain kit (Cat#L34955, Invitrogen) and permeabilized with the IntraPrep kit (Cat# A07803, BeckmanCoulter). The cell suspension was incubated with PHOX2B [Clone B-11] - AlexaFluor® 647 (Cat# SC-376997 AF647, Santa Cruz) and CD44-FITC (Cat# 338804, Biolegend) antibodies diluted at 1/100 during 30 min at 4 °C in dark. Gating strategy is illustrated in Fig. S2A .
Flow cytometry sorting was performed with the SH800 cell sorter (Sony). Cells were detached with TrypLE™ Express Enzyme (Cat# 12604013, Gibco), suspended in PBS and incubated with CD44-FITC antibody diluted at 1/100 during 30 min at 4 °C in dark. DAPI (Cat#62248, ThermoScientific) was used at 1/1000 to exclude dead/dying cells. The first gating for sort was based on FSC/SSC and represents 60% for IC-pPDXC-63 and 75% for SK-N-SH. Doublet cells were eliminated by gating on SSC-W/SSC-H followed by FSC-W/FSC- H. The second gating based on DAPI negative staining eliminated dead/dying cells. The boundaries between positive staining and negative staining were always more than 1 Log of fluorescence intensity (Fig.S2B ). A control tube without staining was always analyzed to determine auto-fluorescence.
Flow cytometry sorting was performed with the SH800 cell sorter (Sony). Cells were detached with TrypLE™ Express Enzyme (Cat# 12604013, Gibco), suspended in PBS and incubated with CD44-FITC antibody diluted at 1/100 during 30 min at 4 °C in dark. DAPI (Cat#62248, ThermoScientific) was used at 1/1000 to exclude dead/dying cells. The first gating for sort was based on FSC/SSC and represents 60% for IC-pPDXC-63 and 75% for SK-N-SH. Doublet cells were eliminated by gating on SSC-W/SSC-H followed by FSC-W/FSC- H. The second gating based on DAPI negative staining eliminated dead/dying cells. The boundaries between positive staining and negative staining were always more than 1 Log of fluorescence intensity (Fig.
10
Yolk Sac Cell Receptor Profiling
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Cell suspensions from dissociated yolk sacs were incubated with indicated specific Abs and then analyzed on a CyAn ADP Analyzer (Beckman Coulter). DAPI‐positive dead cells were excluded from analysis. Cytosolic and PM VEGFR2 staining was performed using an IntraPrep kit (Beckman Coulter), and the data were analyzed using Summit 4.3 software (Beckman Coulter). The following Abs were used: anti‐VEGFR2‐PE‐conjugated (89106); anti‐IgG1‐PE‐conjugated (11711); anti‐CD202b (Tie2)‐PE‐conjugated (Tek4); anti‐IgG1 k isotype‐PE‐conjugated (RTK2071); anti‐FLK1‐APC‐conjugated (Avas12alpha1); anti‐IgG2a k isotype‐APC‐conjugated (R3595); anti‐CD71‐FITC‐conjugated (C2); anti‐IgG1 k isotype‐FITC‐conjugated (R3‐34); anti‐CD117‐PE‐Cy7‐conjugated (2B8); and anti‐IgG2bk isotype‐PE‐Cy7 conjugated.
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