2×105 macrophages were seeded in a 12-well tissue culture plate. After 2 days of terminal differentiation, macrophages were stimulated with 500 ng/mL LPS (Sigma), 3 hours prior to the addition of 2×105 cancer cells (E:T ratio 1:1). 1 hour before the addition of target cells, 1 µg/mL dexamethasone was added. After 4 hours of co-culture, the cells were collected and stained for CD86 expression before flow cytometric analyses were performed using a CytoFLEX S flow cytometer (Beckman Coulter). Prior to the staining procedure, cells were blocked for 5 min at RT with human Fc-Block TruStain FcX (BioLegend), according to the manufacturer’s instructions. Data analysis was performed using FlowJo V.10. Subsequent cytokine analysis was performed using the human tumor necrosis factor alpha (TNF-α) ELISA Ready-Set-Go! Kits (R&D Systems) according to the manufacturer’s instructions.
Fc block human trustain fcx
Manufactured by BioLegend
Sourced in United States
Fc-Block (Human TruStain FcX) is a laboratory reagent designed to block Fc receptors on human cells. It is used to prevent non-specific antibody binding in flow cytometry and other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Cytoflex s flow cytometer, by Beckman Coulter (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ifn α2b, by Miltenyi Biotec (2 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Biosciences facs canto 2, by BD (1 mentions)
Rbc lysis fixation solution, by BioLegend (1 mentions)
Zombie aqua live dead stain, by BioLegend (1 mentions)
Fortessa x20 flow cytometer, by BD (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Fluorochrome conjugated antibodies against, by BD (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Facslyric, by BD (1 mentions)
Neuraminidase, by Roche (1 mentions)
Cd3 apc cy7, by BioLegend (1 mentions)
Hla dr pe cf594, by BD (1 mentions)
Cd14 bb700, by BD (1 mentions)
10 protocols using fc block human trustain fcx
1
Macrophage Activation and Tumor Cytotoxicity
2
Macrophage-Mediated Cancer Cell Cytotoxicity
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2×105 macrophages were seeded in a 12-well tissue culture plate. After 2 days of terminal differentiation, macrophages were stimulated with 500 ng/mL LPS (Sigma), 3 hours prior to the addition of 2×105 CD19+ Raji and 2×105 CD19low K562 cancer cells. After 24 hours and 48 hours of co-culture, the cells were collected and filtered through a 70 µM pore size filter. The cell mixture was stained for CD19-PE expression for 20 min at RT before flow cytometric analyses were performed using a CytoFLEX S flow cytometer (Beckman Coulter). Prior to the staining procedure, cells were blocked for 5 min at RT with human Fc-Block TruStain FcX (BioLegend), according to the manufacturer’s instructions. Data analysis was performed using FlowJo V.10.
3
Flow Cytometric Analysis of Cell Markers
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To detect surface markers and reporter genes, flow cytometric analyses were performed using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, California, USA) with the following antibodies: hCD45-PE-Cy7 (cat. no. 25–0459), hCD11b-APC (17-0118-41), hCD14-PE (12-0149-41), hCD163-APC (17-1639-41), hCD86-PE (12-0869-41), hCD25-Alexa Fluor 488 (53–0259) (all from eBioscience, San Diego, USA) and CD69-Pacific Blue 450 (cat. no. 310920, BioLegend, San Diego, USA). Prior to the staining procedure, cells were blocked for 5 min at room temperature (RT) with human Fc-Block TruStain FcX (BioLegend), according to the manufacturer’s instructions. Data analysis was performed using FlowJo V.10.
4
Flow Cytometry Analysis of mRNA-LNP Biodistribution
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Frozen LN samples were thawed at 37°C and washed twice in warm RPMI 1640 + 10% fetal bovine serum. Samples were then stained with Live/Dead (L/D) FVD780 in PBS for 15 min at 4°C. All of the other staining steps were conducted in fluorescence-activated cell sorting buffer containing PBS (Gibco, Waltham, MA), 0.1% BSA (Sigma-Aldrich, St. Louis, MO), and 2 mM EDTA (Invitrogen, Carlsbad, CA). Cells were washed twice, resuspended in Fc block (Human TruStain FcX, BioLegend, San Diego, CA), and incubated for 15 min at 4°C. After blocking, cells were stained with the surface antibodies listed in Table S1 . Samples were analyzed on an Attune NxT multicolor flow cytometer (Thermo Fisher Scientific). Data were analyzed using FlowJo version X (Tree Star, Ashland, OR).
A Wilcoxon matched-pairs signed rank test was performed to determine significant differences between percentage of EGFP positivity of our mRNA-LNP in the dLN compared with the mesenteric LN across cell types, including macrophages, cDCs, pDCs, classical monocytes, and B and T cells. Gates were set using mesenteric LNs (% EGFP+ cells) samples and fluorescence minus one.Table S2 lists the primary markers for all of the NHP cell populations characterized.
A Wilcoxon matched-pairs signed rank test was performed to determine significant differences between percentage of EGFP positivity of our mRNA-LNP in the dLN compared with the mesenteric LN across cell types, including macrophages, cDCs, pDCs, classical monocytes, and B and T cells. Gates were set using mesenteric LNs (% EGFP+ cells) samples and fluorescence minus one.
5
Multiparametric Immunophenotyping of PBMCs
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PBMCs were isolated, washed in FACS buffer (1% BSA in PBS) and resuspended in 50 µl blocking solution (50 µl FACS buffer, 0,5 µl Fc-Block (Human TruStain FcX, BioLegend, San Diego, CA, USA), 0,5 µl human serum). The cells were incubated for 15 min at 4 °C, washed once in FACS buffer and resuspended in antibody solution containing the fluorescently labelled antibodies IL-6R-APC, TLR2-BV421, CD45-BV510, CD14-APC/Cy7, B220-PE, CD3-AlexaFlour488 and CD56-PE/Cy7. Cells were incubated for 60 min at 4 °C in the dark, centrifuged and washed with FACS buffer. To lyse remaining erythrocytes the cell pellet was resuspended in 1x RBC Lysis/Fixation Solution (BioLegend San Diego, CA, USA) diluted in water and incubated for 15 min at room temperature in the dark. The cells were washed again in FACS buffer, resuspended in 200 µl FACS buffer and analyzed by flow cytometry using the BD Biosciences FACS Canto II (Becton-Dickinson, Heidelberg, Germany).
6
Multiparametric Flow Cytometry of Whole Blood
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Staining was performed on whole blood. 100 µl of blood was washed in PBS and incubated with Zombie Aqua live/dead stain (product number 423101; BioLegend) at room temperature for 10 min. Samples were incubated with FC-Block (Human TruStain FcX, product number 422302; BioLegend) in staining buffer (PBS, 5 mM EDTA, 0.5% BSA), followed by addition of primary antibodies in staining buffer and incubation on ice for 20 min. Cells were washed and incubated with fluorophore-conjugated streptavidin on ice for 15 min. Erythrocytes were lysed with chilled ACK lysis buffer (product number A10492-01; Gibco), and cells were fixed in 4% PFA for 20 min. Cells were resuspended in staining buffer and analysed on a BD X20 Fortessa flow cytometer. Data were analysed using FlowJo (FlowJo, LLC). See Table S2 for flow cytometry reagents.
Table S2
7
Phenotypic analysis of immune cells
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PBMCs, lymphocytes isolated from tissues or purified CD8+ T cells, were labeled using fluorescent mAbs directed against surface molecules (20 min at 4°C), washed in PBS with 0.2% BSA (Sigma-Aldrich), and acquired using FACSVerse or FACSLyric (BD Biosciences, Franklin Lakes, NJ, USA). When required, cells were blocked using FC-block (human Trustain FcX, BioLegend, San Diego, CA, USA), and viability was analyzed using the Zombie NIR or Violet viability kit (BioLegend). Cells were labeled either directly ex vivo or, where indicated, after 30 min of treatment with 25 mU neuraminidase (Roche Diagnostics, Rotkreuz, Switzerland) at 37°C. All mAbs were purchased from BioLegend, with the exception of fluorochrome-conjugated antibodies against CD3, CD8, CLA, CD45RA, LAG3, CXCR3, CCR4, and CCR7 (BD Bioscience); Siglec-9, CCR1 and CCR7 (R&D Systems, Minneapolis, USA); Siglec-7 (Beckman Coulter, Brea, CA, USA), and TNF-α (eBioscience, Waltham, MA, USA). Each mAb was titrated on PBMCs before use.
8
Multiparametric Analysis of IFN-α Signaling
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Thawed cells were labeled with viability dye in PBS for 20 min at 37°C, followed by surface staining for 15 min at room temperature in PBS 2% FBS. After stimulation with IFN-α2b 40.000 IU/ml (Miltenyi Biotec) for 15 min at 37°C, cells were fixed and permeabilized using Cytofix Fixation Buffer (BD Biosciences) pre-warmed to 37°C and Perm Buffer III (BD Biosciences) pre-cooled to −20°C, according to the instructions of the manufacturer. Fc Block (Human TruStain FcX, Biolegend) in PBS was used to prevent the unwanted binding of Fc receptor CD16 antibody. Finally, intracellular staining was performed for 30 min at room temperature in PBS with 0.5% BSA. The following panel was used: Viability Dye Aqua Brilliant Violet 510 (L34957 A+B, Invitrogen), CD3 APC-Cy7 (317341, Biolegend), CD19 APC-eFluor780 (47-0199-42, Invitrogen), CD56 APC-eFluor780 (47-0567-42, eBiosciences), CD14 BB700 (566466, BD Biosciences), CD16 Alexa Fluor 647 (557710, BD Biosciences), HLA-DR PE-CF594 (562304, BD Biosciences), and phospho-STAT1 Brilliant Violet 421 (566238, BD Biosciences).
9
Human FACS Staining Protocol
10
Quantifying Antiviral Protein Levels
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For the analysis of the protein levels of selected ISGs (ISG15, PKR, and MX1), PBMCs were thawed at 37°C, counted, and seeded in a 96-well plate, and then analyzed directly or stimulated overnight at 37°C with or without IFN-α2b 40.000 IU/ml (Miltenyi Biotec). After stimulation, cells were labeled with a viability dye for 30 min at room temperature in PBS, followed by surface staining in PBS 2% FBS for 20 min at 4°C. Cells were then fixed and permeabilized using the FOXP3/Transcription Factor Fixation/Permeabilization Buffers (eBioscience) according to the instructions of the manufacturer. Fc Block (Human TruStain FcX, Biolegend) in PBS was used to prevent the unwanted binding of Fc receptor CD16 antibody. Finally, intracellular staining was performed for 30 min at room temperature in Permeabilization Buffer (eBioscience). The flow cytometry panel was the following: Viability Dye eFluor780 (65-0865-14, Invitrogen), CD14 Brilliant Violet 421 (563743, BD Biosciences), MXA Alexa Fluor 488 (AMab237298, Abcam), PKR Alexa Fluor 647 (AMab224921, Abcam), and ISG15 PE (IC8044P, R&D Systems).
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