After the treatment, cells were collected and washed twice with ice cold PBS and fixed with 4% paraformaldehyde in ice cold PBS for 5 min. Cells were centrifuged at 1200 rpm for 5 min and washed again in ice cold PBS. Then cells were incubated for 30 min at 4 °C with staining solution (mix per sample: 200 μL ice cold PBS + 4 μL Fetal Bovine Serum + 1 μL Anti-CRT antibody #FMC75 [Abcam Cambridge, MA]). Isotype-control IgG1 (BD Biosciences; CA, USA) was used as control (mix per sample: 200 μL ice cold PBS + 4 μL Fetal Bovine Serum + 1 μL Isotype-control IgG1). Next, cells were washed twice with ice cold PBS, centrifuged for 5 min at 1200 rpm and resuspended in ice cold PBS. Samples were analyzed by flow cytometry (Attune-AB Applied Biosystems) to identify the percentage of CRT positive cells (i.e. cells that externalized the CRT) and also the intensity of CRT. We included a positive control based on the treatment of HCT116 colorectal cancer cells treated with Oxaliplatin [30 (link)].
Igg1 isotype control
Manufactured by BD
Sourced in United States
The IgG1 isotype control is a laboratory reagent used as a reference in immunoassays and flow cytometry experiments. It serves as a control to establish a baseline response and distinguish specific from non-specific binding of antibodies. The IgG1 isotype control has the same immunoglobulin subclass and species of origin as the test antibody, but does not recognize any known antigen.
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Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Sy 3 8, by Thermo Fisher Scientific (2 mentions)
Percp conjugated anti cd45, by BD (2 mentions)
L mimosine, by Merck Group (2 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (2 mentions)
Anti mouse alexa488, by Abcam (2 mentions)
Function blocking anti human icam 1 15.2, by Bio-Rad (2 mentions)
Anti cd11b cd18 mab cbrm1 29, by Cell Signaling Technology (2 mentions)
Rabbit anti akt and anti β catenin phospho forms, by Cell Signaling Technology (2 mentions)
Anti human mhc class 1 1 b 548, by Abcam (2 mentions)
Hrp conjugated anti mouse and anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Dmem including media supplements, by Merck Group (2 mentions)
Rabbit anti ki67, by Abcam (2 mentions)
Akt inhibitor 8, by Merck Group (2 mentions)
V bottom 96 well plate, by Corning (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Anti cxcr4, by BD (1 mentions)
Pe mouse igg1, by BD (1 mentions)
12 protocols using igg1 isotype control
1
Flow Cytometric Analysis of CRT Exposure
2
Quantifying DENV Internalization in Raji-DC-SIGN Cells
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The amount of DENV internalization by host cells was determined in the Raji B-cell leukemic cell line stably expressing the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) molecule [27 (link)]. Briefly, 50 µL/well of live and psoralen-inactivated DENV at a concentration of 1.7 × 107 pfu/mL were added to a V-bottom 96-well plate (Corning Incorporated, NY, USA) containing 1 × 105 per 50 µL of Raji-DC-SIGN cells. After 4 h of incubation in a humidified 37 °C CO2 incubator, the cells were washed, fixed, and permeabilized using the BD Cytofix/Cytoperm™ kit (BD Biosciences, San Jose, CA, USA) following the manufacturer’s instructions. The fixed cells were stained with a monoclonal antibody to DENV envelope protein (3H5) or an IgG1 isotype control (BD Biosciences) followed by a FITC (fluorescein isothiocyanate) labeled secondary goat-anti-mouse IgG antibody (NBACC). Cells not treated with virus served as the negative control. After 30 min of staining for each step, the cells were sorted on a FACSCANTO II (BD Biosciences). The percent of 3H5 positive cells represented the number of cells that captured and internalized DENV using the DC-SIGN receptor.
3
FACS Analysis of SAP Protein Expression
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FACS was used to quantify SAP protein expression encoded by SH2D1A as previously described72 . In brief, blood cells were incubated with mouse anti-human CD3 (BD Bioscience, Clone SK7, cat 345767), mouse anti-human CD8-PerCP (BD Bioscience, Clone SK1, cat 345774), and mouse anti-human CD56-PE (BD Bioscience, Clone MY31, cat 345810). Samples were then fixed, washed, an permeabilized. The anti-SAP antibody (Stratech Scientific Biosciences; clone 1C9, cat H00004068-MO1) or isotype control antibody (IgG1 isotype control; BD Biosciences 349040). Samples were again washed and stained with anti-mouse IgG1-FITC (Dako; F0479) before FACS analysis.
4
Siglec-1-Mediated HIV-1 VLP Uptake
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Uptake experiment were performed as previously described (Izquierdo-Useros et al., 2012a (link); Izquierdo-Useros et al., 2014 (link); Pino et al., 2015 (link)) using p24Gag HIV-1Gag−eGFP VLP (GFP VLP). Briefly, monocytes transfected (or not) with control siRNA, or with siRNA directed against Siglec-1, and differentiated for 3 days in cmCTR or cmMTB, were washed once with PBS prior to addition of 2 ng/mL of GFP VLP. Binding was performed during 3.5 hr at 37°C in a 5% CO2 incubator. Cells were then detached with cell dissociation buffer (Gibco) and prepared for flow cytometry analysis on a BD LSRFortessa (TRI-Genotoul platerform). Same experiment was also performed blocking monocyte-derived macrophages at RT for 15 min with 10 μg/mL of mAb α-Siglec-1 7–239 (Abcam), IgG1 isotype control (BD Biosciences) or leaving cells untreated before VLP addition.
5
Multicolor Flow Cytometry Analysis of Immune Cells
6
Isolation and Phenotyping of iNKT Cells
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Heparinized blood was layered over an equal volume of Lymphoprep™ (Axis-Shield PoC AS, Oslo, Norway) solution, which was centrifuged at 2,000 rpm for 30 minutes. Peripheral blood mononuclear cells (PBMCs) were isolated from the interface and used to determine the number of iNKT cells, as described previously.12 (link)
PBMCs were incubated at 4℃ for 30 minutes with fluorochrome-conjugated monoclonal antibodies, including PE-6B11 (BD Biosciences, San Jose, CA, USA) or PE-anti-TCRvα24 (Beckman Coulter, Marseille, France) and APC-anti-TCRvβ11 (BD Biosciences). PE-mouse IgG1 (BD Biosciences) and APC-mouse IgG1 (BD Biosciences) were used for isotype control antibodies. For intracellular staining of iNKT cells, fixation and permeabilization were performed using Cytofix/Cytoperm kits (eBiosciences) according to the manufacturer's instructions. These cells were incubated with FITC-IL-4 mAb (BD Biosciences), FITC-IFN-γ mAb (BD Biosciences), or FITC-IL-10 mAb (BD Biosciences). IgG1 isotype control (BD Biosciences) was used. After gating the lymphocytes, the frequency of Vα24+Vβ11+ or 6B11+Vβ11+ iNKT cells was expressed as a percentage of lymphocytes. Intracellular IL-4+, IFN-γ+ or IL-10+ cells were presented as a percentage of Vα24+Vβ11+ or 6B11+Vβ11+ iNKT cells (Fig. 1 ).
PBMCs were incubated at 4℃ for 30 minutes with fluorochrome-conjugated monoclonal antibodies, including PE-6B11 (BD Biosciences, San Jose, CA, USA) or PE-anti-TCRvα24 (Beckman Coulter, Marseille, France) and APC-anti-TCRvβ11 (BD Biosciences). PE-mouse IgG1 (BD Biosciences) and APC-mouse IgG1 (BD Biosciences) were used for isotype control antibodies. For intracellular staining of iNKT cells, fixation and permeabilization were performed using Cytofix/Cytoperm kits (eBiosciences) according to the manufacturer's instructions. These cells were incubated with FITC-IL-4 mAb (BD Biosciences), FITC-IFN-γ mAb (BD Biosciences), or FITC-IL-10 mAb (BD Biosciences). IgG1 isotype control (BD Biosciences) was used. After gating the lymphocytes, the frequency of Vα24+Vβ11+ or 6B11+Vβ11+ iNKT cells was expressed as a percentage of lymphocytes. Intracellular IL-4+, IFN-γ+ or IL-10+ cells were presented as a percentage of Vα24+Vβ11+ or 6B11+Vβ11+ iNKT cells (
7
Tim-3 Blockade for Functional Assays
8
Quantification of Circulating Progenitor Cells
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Circulating CD34+/CD133+/CD45low cells, including EPCs were measured by flow cytometry using minor modifications of a previously described method [16 (link)–18 (link)]. In brief, EDTA-treated peripheral blood was incubated with the test or control reagent. The reagent mixture consisted of nucleic acid dye (SY-III-8; Molecular Probe, Eugene, OR, USA), peridinine chlorophil protein (PerCP)-conjugated anti-CD45 (Becton Dickinson, San Jose, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (Becton Dickinson) and phycoerythrin (PE)-conjugated anti-CD133 (Miltennyi Biotec, Bergisch Gladbach, Germany). Isotype controls were used as the negative controls based on the species and immunoglobulin (Ig) G control antibodies (IgG1 isotype control; Becton Dickinson). Flow cytometric analysis was then performed using a FACS Calibur laser flow cytometer (Becton Dickinson) according to the manufacturer’s instructions. Each measurement consisted of 106 events of all white blood cells, which exceeded the threshold set for SY-III-8 fluorescence (nucleated cells). The absolute number of CD34+/CD133+/CD45low cells per mL was calculated based on the cells-to-the whole-blood cell count.
9
Multicolor Flow Cytometry Analysis of NK Cells
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For measurement of phosphorylated pSTAT-4 20 min and for measurement of IFN-γ and perforin 18 h in vitro stimulated PBMC (1x106/ml) from HC and MM patients were analyzed. Three hours before IFN-γ analysis, brefeldin A was added at concentration of 10 μg/ml. The cells were then stained with mAbs against cell surface molecules: CD3PerCP, CD56PE (for IFN-γ analysis) and CD56FITC (for pSTAT-4 and perforin analyses) (Becton Dickinson), followed by fixing and permeabilizing with the Permeabilizing Solution 2 (Perm 2) (Becton Dickinson) accords to the manufacturer’s instructions. Then the cells were incubated with anti-IFN-γFITC (clone 25723.11), anti-pSTAT-4PE (clone 38/p-Stata4) (Becton Dickinson), anti-Perforin RPE (clone deltaG9) (Invitrogen, Madison, USA) or IgG1 isotype control (Becton Dickinson). Finally, the cells were washed and analyzed by flow cytometry directly after preparation. The percentages and MFI of intracellular IFN-γ, pSTAT-4 and perforin were analyzed in CD3−CD56+ NK cells and their CD3−CD56dim+ and CD3−CD56bright+ subsets.
10
Quantifying Circulating Endothelial Progenitor Cells
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We measured circulating CD34 + CD133 + CD45low cells, which include EPCs, using flow cytometry based on a previously described method [7] (link), [8] (link), [9] (link) with minor modifications. In brief, the reagent mixture consisted of a nucleic acid dye (SY-III-8; Molecular Probe), a peridinine chlorophil protein (PerCP)-conjugated anti-CD45 (Becton Dickinson), a fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (Becton Dickinson) and a phycoerythrin (PE)-conjugated anti-CD133 (Miltennyi Biotec). Isotype controls were used as negative controls based on the species and immunoglobulin (Ig) G control antibodies (IgG1 isotype control; Becton Dickinson). Flow cytometric analysis was performed using the FACS Calibur laser flow cytometer (Becton Dickinson) according to the manufacturer's instructions (Supplemental file 1). The absolute number of CD34 + CD133 + CD45low cells per milliliter was calculated based on the cells-to-the white blood cell count. To minimize any methodological variations, each sample was analyzed with two independent experiments, and the mean value was calculated.
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