For pinnae,1×106 cells were incubated in an Fc receptor block (anti-CD16) for 10 min and stained with live/dead stain-aqua (Invitrogen) (1:5000), F4/80-PE/Cy7 (1:200), CD45-alexaflour700 (1:200) and Gr1-PE (1:200), or Ly6C & Ly6G, or CD3-FITC for 30 min in ice in the dark. Samples were washed thrice in FACS buffer and analyzed with a Cytek Aurora cytometer. For peritoneal cells, 1×106 cells were incubated in an Fc receptor block (anti-CD16) for 10 min and stained with CD45R, CD11c, CD11b, F4/80, Ly6C, Ly6G, and live/dead stain for 30 min in ice. Samples were washed thrice in FACS buffer and analyzed with a Cytek Aurora cytometer. All flow cytometry data were analyzed with FlowJo software (Tree Star, USA).
Aurora cytometer
Manufactured by Cytek
Sourced in United States
The Cytek Aurora is a flow cytometer designed for high-parameter and high-throughput analysis of single cells. It features a modular architecture that allows for the simultaneous detection of up to 40 parameters per cell. The Aurora utilizes Cytek's proprietary technology to provide efficient excitation and detection of fluorescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Zombie nir live dead exclusion dye, by BioLegend (3 mentions)
Live dead fixable blue dye, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Lonza (3 mentions)
Live dead near ir stain, by Thermo Fisher Scientific (2 mentions)
Formaldehyde, by Thermo Fisher Scientific (2 mentions)
Lsr 2, by Cytek (2 mentions)
Rpa t8, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Human trustain fcx, by BioLegend (2 mentions)
Novablock, by Thermo Fisher Scientific (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Cd206, by BioLegend (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Foxp3 fix perm kit, by Thermo Fisher Scientific (2 mentions)
Spectroflo, by Cytek (2 mentions)
Spectroflo software, by Cytek (2 mentions)
Aqua live dead stain, by Thermo Fisher Scientific (1 mentions)
Aria 3, by BD (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Collagenase a, by Roche (1 mentions)
41 protocols using aurora cytometer
1
Multicolor Flow Cytometry of Immune Cells
2
Characterizing Immune Responses in SARS-CoV-2 Infection
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SARS-CoV-2 MA10 virus-infected mice were anesthetized at day 4 post infection. Mouse lung and spleen were collected and minced in RPMI and filtered through a 70 μm filter, then washed and resuspended in Red blood cell lysis buffer, then resuspendend in MACS buffer. Isolated lung and spleen mononuclear cells were stained with anti-CD64 BV711, anti-CD11b PE, anti-CD45.2, PerCP-Cy5.5, anti-Ly6G FITC, anti-MHCII PE-Cy7, anti-Ly6C APC, anti-Siglec-F AF700, and anti-F4/80 APC-Cy7. The stained cells were washed and resuspended in 4% PFA for 30 minutes. Cells were acquired on a Cytek Aurora cytometer. Flow cytometry analysis was done with the FlowJo software.
3
Cryopreserved PBMC Cytometry Analysis
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Cryopreserved PBMCs were used, and all samples were acquired on an Aurora cytometer (Cytek Biosciences) and analyzed with Flowjo software, version 10 (TreeStar).
4
Flow Cytometry Cell Purity Assay
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Primary cells were stained for purity post-isolation using anti-CD45 (HI30, BD), anti-CD8 (RPA-T8, BD), and Near IR Live/dead stain (Invitrogen), then fixed with 4% formaldehyde (Thermo) and analyzed using a BD LSR II or a Cytek Aurora cytometer. Data were analyzed using FlowJo 10 software.
5
Comprehensive Flow Cytometry Analyses
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Splenocytes and bone marrow cells were stained with fluorescently labeled antibodies, and different subsets of cells were gated as shown in Fig. S1 F . Cells derived from the transferred B1-8hi cells were further gated based on their different fluorescent protein expression from the host or their binding to NP or NIP. In polyclonal response against dextran, splenocytes and bone marrow cells were stained with 10 µg/ml dextran-FITC to identify antigen-specific cells in each population. Intracellular staining were used to identified NP- or dextran-specific PCs at later time points (days 7, 19, and 100) of the responses, as these cells have lost their surface BCR expression. Data were acquired using an Aria III or LSRII flow cytometer (BD) or an Aurora cytometer (Cytek).
6
Isolation of Lung Mononuclear Cells
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Vehicle– or diABZI-4–treated mice were anesthetized with ketamine and transcardially perfused with PBS until lung tissue turned white in color. Lung tissue was minced and digested in collagenase A (Roche) and 30 μg/ml DNase I (Sigma-Aldrich) in RPMI at 37°C for 45 min. Digested tissue was filtered through a 70 μm filter and washed and resuspended in MACS buffer. Isolated lung mononuclear cells were stained with anti-CD45.2 BV650, anti-CD11b BV510, anti-Ly6C APC, anti-TCRß PerCP-Cy5.5, anti-CD8 Alexa 700, anti-CD11c eF450, anti-CD64 BV711, anti-Ly6G PE-Cy7, anti-Ly6G FITC, anti-CD3 PerCP-ef710, anti-CD4 APC-Cy7, anti-TCRẟ PE, anti-B220 PE-Cy5, anti-CD326 APC, anti-CD45.1 APC-Cy7. Cells were acquired on a Cytek Aurora cytometer. Flow cytometry analysis was done with the FlowJo software.
7
Immune Profiling of 4T1 Tumor Cells
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4T1 tumors were resected, minced, and digested in Liberase TL (Roche, Basel, Switzerland) and DNAse (Roche, Basel, Switzerland) for 30 min at 37 °C under constant rotation. Single cell suspension was filtered through 70 μm strainers and washed with PBS. Cells were then incubated with Live Dead NIR in PBS for 20 min and then Fc blocking antibody (2.4G2, Biolegend, San Diego, CA, USA) in FACS buffer at 4 °C for 15 min. Fluorochrome-conjugated antibodies specific to cell surface markers were added and incubated for 45 min at 4 °C. Fluorochrome-conjugated antibodies to CD45 (1:500 FITC clone 30-F11), CD3 (1:100 BV785 clone 17A2), CD8 (1:300 APC clone 53-6.7), CD4 (1:300 BB700 clone RM4-5), MHC-II (1:200 BV605 clone M5/114.15.2), and CD11c (1:200 AF700 clone N418) were obtained from BioLegend (San Diego, CA, USA). The stained samples were acquired in an Aurora cytometer (Cytek, Fremont, CA, USA) and data was analyzed using FlowJo version 10.8 (FlowJo LLC, Ashland, OR, USA).
8
Functional Analysis of T-bet Variants
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This experiment was performed as previously described (Yang et al., 2020 (link)). Briefly, CD4+ T cells were isolated from PBMCs from five healthy donors (controls), the T-bet WT/M heterozygous father (TBX21 WT/M), and the T-bet M/M P (TBX21 M/M), with anti-CD4 Ab-coated beads (Miltenyi Biotec). This population of cells was then expanded with anti-CD3/CD28 Ab-coated Dynabeads (Thermo Fisher Scientific) at a 1:1 (cell:bead) ratio under Th0 cell–polarizing conditions, in the presence of 100 IU/ml proleukin IL-2 (PROMETHEUS) and anti-human IL-10 neutralizing Ab (Thermo Fisher Scientific). Cells were restimulated with anti-CD3/CD28 Ab-coated Dynabeads every 7–8 d. P’s CD4+ T cells were transduced with retroviruses obtained from pLZRS-ires-ΔCD271 plasmids with or without WT TBX21 or EV cDNA, as previously described (Martínez-Barricarte et al., 2016 (link); Yang et al., 2020 (link)). Cells were stained with Zombie NIR Live-Dead exclusion dye (BioLegend) and anti-CD271-FITC Ab (BioLegend) and were then fixed and permeabilized. They were stained with anti-TNF-α-BV510, anti-IL-13-APC, anti-IL-4-PE, anti-IL-17A-BV785, anti-IL-10-PE-Dazzle594, anti-IL-5-BV421, and anti-IFN-γ-PerCp-Cy5.5 Abs and subjected to flow cytometry in an Aurora cytometer (Cytek).
9
RBD-Streptavidin Tetramer Flow Cytometry
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Recombinant biotinylated RBD (BioLegend) was reacted with PE-Streptavidin (BioLegend) in a 4:1 molar ratio at 4°C for 2 hours. For flow cytometry studies, PBMCs underwent Fc-blockade with Human TruStain FcX (BioLegend), NovaBlock (Thermo Fisher Scientific), APC-Streptavidin (BioLegend), and mouse phycoerythrin (PE)-IgG2b isotype control (BioLegend) for 10 min at room temperature, followed by allophycocyanin (APC) anti-PE (BioLegend) for 10 min. Then cells were washed twice and stained with the RBD tetramer and antibodies against other surface proteins for 20 min at room temperature in the dark, followed by resuspension in 1% paraformaldehyde and acquisition on a 5-laser Aurora cytometer (Cytek Biosciences).
10
Multicolor Flow Cytometry Analysis of Neutrophil Surface Markers
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Neutrophils were fixed with 4% PFA for 30 min at 4 °C, washed and stained for 20 min in the dark with the following anti-human antibodies: CD45-APC-Cy7 (clone 2D1; Biolegend, San Diego, CA, USA), CD54-BV421 (clone HA58; Biolegend), CD62L-BV711 (clone SK11; Biolegend), CXCR4-BV-785 (clone 12G5; Biolegend), CD66b-APC (clone REA306; Miltenyi Biotec), CD15-FITC (Miltenyi Biotec) and CD16-PE (clone 3G8; BD Biosciences, Franklin Lakes, NJ, USA). A live/dead fixable blue dead cell stain kit (Thermo Scientific; Waltham, MA, USA) was used to assess cell death in cultures before fixation. Fluorescence Minus One (FMO) controls were used to identify and gate positive populations (Figure 1 and Figure S1b ). Analysis was performed on an LSRII flow cytometer (BD; Ashland, Wilmington, DE, USA) or Aurora cytometer (Cytek Biosciences; Fremont, CA, USA) and assessed using FlowJo software (BD) or OMIQ (www.omiq.ai (accessed on 13 July 2021)). The expression of surface markers was measured by the percentage of positive cells.
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