Bone marrow (BM) cells were obtained after BM was flushed, treated with red blood cell lysis buffer (150 mM NH4CL and 10 mM KHCO3) and washed with staining media (Hank’s Buffered Salt Solution supplemented with 2% fetal bovine serum)39 (link). 8 × 106 unfractionated BM cells were stained with unconjugated rat lineage-specific antibodies (Ter119, Mac1, Gr-1, B220, CD5, CD3, CD4, CD8) followed by staining with goat anti-rat PE-Cy5 antibody. Cells were then stained using c-Kit-APC-eFluor780, Sca1-PB, CD48-BV711, CD150-PE, Flt3-Biotin, FcgR-PerCP-eFluor710, CD34-FITC, CD41-BV605, and CD105-APC antibodies. Secondary staining was performed with streptavidin-PE-Cy7. Zombie NIR fixable viability kit (BioLegend) was used for dead cell exclusion. Data were collected on a 5 laser Aurora spectral flow cytometer (Cytek Biosciences). Data analysis was performed using FlowJo (BD biosciences) software. Further antibody details are listed in Supplementary Table 5 .
5 laser aurora spectral flow cytometer
Manufactured by Cytek
Sourced in United States
The 5 laser Aurora spectral flow cytometer is a research-grade instrument designed for high-performance multiparameter analysis. It utilizes spectral detection technology to capture a full emission spectrum for each particle, enabling advanced data analysis and greater insights into complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Zombie nir fixable viability kit, by BioLegend (1 mentions)
Pharm lyse, by BD (1 mentions)
Formaldehyde solution, by Merck Group (1 mentions)
Ficoll gradient, by Eurobio Scientific (1 mentions)
Golgistop, by BD (1 mentions)
8 protocols using 5 laser aurora spectral flow cytometer
1
Isolation and Immunophenotyping of Murine Hematopoietic Stem Cells
2
High-Dimensional Flow Cytometry of PBMCs
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PBMC from patients and controls of the Rotterdam cohort were stained with a 40-color antibody panel and collected using a 5 laser Aurora spectral flow cytometer (Cytek Biosciences, CA). All samples were stained as described previously46 (link) with the adaptation of including annexin V to exclude dead cells and all buffers contained all buffers contained 2.5 mM CaCl2. Cleaning of flow data was performed in SpectroFlo (version 2.2.0) (Supplementary Fig. S4 ). The unsupervised, and statistical inference portions of the flow cytometry analysis were performed using OMIQ data analysis software(www.omiq.ai ). We used the unsupervised analysis methods based on surface markers without any 2D gating we previously employed46 (link). The workflow included running flowCut to check for changes in channels over acquisition time, UMAP for dimensionality reduction, flowSOM for clustering, and edgeR for statistical inference. For the statistical comparisons of abundance, the Flow Cytometry Standard (FCS) files were subsampled to ensure the same number of events were included per group (either immunotype or disease state).
3
Cryopreserved PBMC Immunophenotyping
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Cryopreserved peripheral blood mononuclear cells were thawed rapidly and incubated in RPMI 1640 medium containing 10% FBS at 37°C for a 3 hour resting period. Peripheral blood mononuclear cells were then washed twice in PBS and incubated in Fc block and True Stain Monocyte Blocker for 5 minutes. Cells were stained with surface antibodies for 15 minutes at 4°C and intracellular antibodies for 30 minutes at 4°C with Brilliant Buffer Plus (eBioscience), staining intracellularly after fixing and permeabilizing the cells in FoxP3 IC fixation buffer (eBiosciences). And the detailed information (including fluorophore, manufacturer, clone, and catalog number) of antibodies used is listed in Table S1, http://links.lww.com/HC9/A336 ). Cells were analyzed on a 5-laser Aurora spectral flow cytometer (Cytek Biosciences, CA, USA) and later analyzed using FlowJo software version 10.8.1.
Data were loaded into R software and subjected to quality control using FlowAI with standard configuration. Live CD3+ CD8+ T cells were gated on good events in FlowJo, downsampled to the same cells using the DownSample plugin for each sample, and concatenated. The UMAP plugin in FlowJO was used to calculate the UMAP coordinates of the resulting cells. FlowSOM was then run on the coordinates of the UMAP map to generate the FlowSOM metaclusters overlaid on the UMAP map.
Data were loaded into R software and subjected to quality control using FlowAI with standard configuration. Live CD3+ CD8+ T cells were gated on good events in FlowJo, downsampled to the same cells using the DownSample plugin for each sample, and concatenated. The UMAP plugin in FlowJO was used to calculate the UMAP coordinates of the resulting cells. FlowSOM was then run on the coordinates of the UMAP map to generate the FlowSOM metaclusters overlaid on the UMAP map.
4
Multiparametric Phenotypic Profiling of PBMCs
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A combination of 20-fluorochrome-conjugated antibodies was designed to investigate the activation status of PBMCs after co-culture. Samples were processed and stained according to the standardized EuroFlow protocols www.euroflow.org/protocols .35 (link) All incubations were performed in the dark. Briefly, the cells of the co-culture were stained with the antibody panel for 30 min at room temperature, washed with PBS and incubated with a viability marker (LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation, Thermo Fisher Scientific) for 30 min at 4°C. Subsequently, cells were incubated in 1x BD Pharm lyse (BD Biosciences) for 10 min and then washed with PBS containing 0.5% bovine serum albumin and 0.1% sodium azide (FACs buffer). The samples were then incubated in PBS with 1% formaldehyde solution (Sigma-Aldrich, #1004965000) overnight. Before acquisition, cells were washed twice with PBS and subsequently measured with 5-Laser Aurora spectral flow cytometer (Cytek Biosciences). Data analysis was performed with Infinicyt software 2.0.6 (Cytognos S.L., Salamanca, Spain).
5
Multiparameter Flow Cytometry of PBMCs
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PBMCs were isolated using a standard Ficoll gradient according to the manufacturer's instructions (Eurobio) and frozen. After thawing, PBMCs (1.106 cells) were stained with the 2 T-cell panels: panel 1 with 15 extracellular markers and panel 2 with 14 extra-and intracellular markers listed in eTable 1A, links.lww.com/NXI/A802 . The viability staining with the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Invitrogen) was performed at 4°C for 20 minutes. Then, the staining with the 2 panels of antibodies was performed at 4°C in staining buffer. The antibodies were added in sequential steps. First, C-C chemokine receptor type 7 (CCR7) was stained for 10 minutes, and then, other antibodies targeting chemokines were added for 10 minutes. Finally, extracellular markers were stained for 20 minutes. For panel 2, a permeabilization and fixation step (eBioscience FoxP3/TF staining buffer set) was performed after extracellular staining for 45 minutes at room temperature, and then, intracellular markers were stained for 30 minutes at room temperature. PBMCs were analyzed at baseline, 24 weeks, and 48 weeks of OCR treatment, using an Aurora 5-laser spectral flow cytometer (Cytek Biosciences). After individual titration of each antibody, a deconvolution matrix was set up, and a quality control procedure was performed prior to each data acquisition according to Cytek recommendations.
6
Cytotoxicity Assay of Neuroblastoma Cells
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Neuroblastoma cells (SK-N-AS control, SK-N-AS shYAP1, and shYAP2) were co-cultured with γδ T cells at an E:T ratio of 1:1 as described in the flow cytometry-based cytotoxicity assay protocol above. CD107a-PE Cy7 antibody was added to each well 30 minutes after the cytotoxic assay was started. GolgiStop (BD Biosciences) was added one hour later at a final concentration of 0.7uL/mL. At the endpoint, γδ T cells were harvested and stained with CD3-BV421, CD56-APC-R700, and γδ TCR-PE, washed twice in FACs buffer and resuspended for data acquisition on the Cytek Aurora 5-laser spectral flow cytometer. See Supplemental Table S2 for antibody information. Data were analyzed using FlowJo version 10.8.1.
7
Isolation and Characterization of Implant-Associated Cells
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After 7 and 21 days after implantation, animals were euthanized with carbon dioxide. For SQ implants, the skin was excised around the implant area, and the implant with the associated capsule was examined away from the dermis. For VML implants, the quadriceps with ECM material was dissected out along the femur to the hip. The skin and abdominal wall were dissected carefully for IP implants to prevent bleeding. The IP cavity was lavaged with 1 mL of serum-free RPMI, and then any visible ECM was removed from the IP cavity. The resulting tissue isolates were digested in 0.25 mg/ml Liberase TM with 0.2 mg/ml DNase I in serum-free RPMI for 45 minutes at 37°C on a shaker at 100 rpm at a total volume of 5 mL per 50 μl implant. Digested samples were filtered through a 70 μm cell strainer and rinsed with room temperature 1xPBS. Samples were spun down at 350xg at room temperature for 5 minutes, and the resulting pellet was washed twice in 1xPBS before staining for 30 minutes in LIVE/DEAD Blue viability dye or 7-AAD. As previously described, the cell pellet was washed three times in cold 1xPBS before staining in a surface antibody cocktail (Supplemental Tables 1 and 2 ,[19 –21 ]). Samples were washed three times and then run on a Cytek Aurora 5 laser spectral flow cytometer. Single color controls for unmixing were made using peripheral blood mononuclear cells.
8
Comprehensive PBMC Functional Profiling
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Two million PBMCs were either directly stained with the “T cell panel” or stimulated with PMA (50 ng/mL) and calcium ionophore (500 ng/mL) for 3.5 h in the presence of brefeldin A (5 µg/mL) before being stained with the “functional panel” (i.e., IL-10, IL-13, IL-17, IFNγ, TNFα). PBMCs were analyzed using an Aurora 5-laser spectral flow cytometer (Cytek). After individual titration of each antibody, a deconvolution matrix was set up according to Cytek recommendations. Quality control procedure was performed prior each data acquisition according to Cytek recommendations.
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