CYNK-001 cells with NK cell purity of ≥90% CD56+CD3-, were used in this assay. Frozen CYNK-001 cells were thawed and washed in staining buffer, phosphate buffered saline (PBS) (10,010–023, Gibco) containing 10% FBS (10,082–147, Gibco). 1 × 106 CYNK-001 cells were stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain (L34966, Invitrogen) in PBS and then blocked with a solution containing Purified Mouse IgG2a, κ Isotype Control (555,571, BD), Human BD Fc Block™ (564,219, BD) and BD Horizon™ Brilliant Stain Buffer (563,794, BD). Fluorophore-conjugated antibodies from BD, Miltenyi Biotec and Biolegend were diluted in staining buffer according to manufacturers’ instructions. The following antibodies were used: CD226 (Clone: D×11 559,789, BD), CD337 (Clone: p30–15 563,385, BD), CD335 (Clone: 9-E2 563,230, BD), CD56 (Clone: 5.1H11 362,510, BioLegend), CD3 (Clone: SK7 560,176, BD), CD14 (Clone: MφP9, 641,394, BD), CD19 (Clone: SJ25C1 641,395, BD), CD336 (Clone: p44–8 744,300, BD), CD314 (Clone: BAT221 130-092-673, Miltenyi Biotec). Samples were acquired on Cytek® Aurora flow cytometer (Cytek, CA, US) and data analyzed on FlowJo™ Software (BD, Version 10.7.2). Two independent experiments had been performed from the six CYNK-001 donors.
Aurora flow cytometer
Manufactured by Cytek
Sourced in United States
The Cytek Aurora flow cytometer is a versatile instrument designed for high-performance multi-parameter analysis of cells and other particles. It features advanced optics and detection capabilities to provide researchers with accurate and reliable data.
Automatically generated - may contain errors
Lab products found in correlation
Spectroflo software, by Cytek (5 mentions)
Zombie nir fixable viability dye, by BioLegend (4 mentions)
Anti cd19, by BioLegend (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Horizon brilliant stain buffer, by BD (3 mentions)
Accutase, by Corning (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Brilliant stain buffer, by BD (2 mentions)
Zombie nir, by BioLegend (2 mentions)
Trustain fcx, by BioLegend (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Zombie aqua viability dye, by BioLegend (2 mentions)
Foxp3 transcription factor staining buffer set, by BD (2 mentions)
Anti cd3 buv395, by BD (2 mentions)
Anti cd161 pe dazzle 594, by BioLegend (2 mentions)
Anti granzyme b alexa fluor 700, by BioLegend (2 mentions)
Anti tnf α efluor 450, by Thermo Fisher Scientific (2 mentions)
114 protocols using aurora flow cytometer
1
Phenotypic Analysis of CYNK-001 NK Cells
2
Comprehensive Multicolor Flow Cytometry Staining Protocol
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Cells were harvested and washed twice in PBS (21-040-CV, Corning) and stained with Zombie NIR (423106, BioLegend) for 15 minutes at room temperature. Cells were washed 3 times in 200 mL of FACS buffer (PBS without Ca2+ or Mg2+ supplemented with 10% FBS) and stained with fluorophore-conjugated antibodies prepared in a mixture of FACS buffer and Brilliant Stain Buffer (566349, BD Biosciences) for 30 minutes at 4°C. Samples were washed 3 times, fixed in 2% paraformaldehyde for 20 minutes at room temperature, and washed 3 times before analysis. For transcription factor or p24 staining, surface-stained and fixed cells were permeabilized in 0.1% Triton X-100 for 5 minutes at room temperature. Antibodies are shown in Supplemental Table 8 . Samples were acquired on a Cytek Aurora flow cytometer (Cytek Biosciences) and analyzed with FlowJo (FlowJo LLC).
3
Testicular Cell Cycle Analysis
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Aliquots of testis single cell suspensions (1×106 cells/ml) were first incubated with Trypan Blue to identify percentages of dead cells (Strober, 2015 (link)). The rest of the suspension was immediately fixed in ice-cold 70% ethanol and stored at −20°C. On the day of the experiment, ethanol-suspended cells were washed twice in 1×PBS and finally resuspended in 300 µl of 20 µg/ml propidium iodide (PI)/Triton X-100 staining solution with 5 µg/ml RNase A at room temperature for 30 min. DNA content measurements were performed using a 5-laser Cytek Aurora flow cytometer equipped with a 50 mW 561 laser. For PI emission, the YG4 band pass (661/18) was used. Flow rate was set to low to achieve the best quality, which was evident from the very low coefficient of variation (CV) values of the peaks (indicating peaks were generated from cells with the same DNA content). Data were analyzed using a classical linear scale. Testicular single cell suspensions from adult wild-type CD-1 testes were used for calibration and as positive controls.
4
Flow cytometry analysis of cardiac macrophages
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For flow cytometry analysis, hearts were perfused via the left ventricular cavity with 10ml of PBS supplemented with heparin (50I.E./ml), immediately mechanically disrupted in digestion buffer (450u/ml collagenase I (Sigma Aldrich, St. Loius, MO, USA), 125u/ml collagenase XI (Sigma Aldrich), 60u/ml hyaluronidase (Sigma Aldrich) and 60u/ml DNAse I (Thermo Fisher Scientific, Waltham, MA, USA) in HBSS) and digested for 1 hour at 30°C. The single cell suspension was stained with a cocktail of antibodies including CD45, CD11b, CD64, CCR2 and TIMD4 (an antibody list including clones, fluorescent dyes and manufacturer data can be found in the supplementary material). Fluorescence minus one controls (FMO) for each marker were used to assure correct compensation. The Zombie UV fixable kit (Biolegend, San Diego, CA, USA) was used to assure cell viability. Stained cells were analyzed with a Cytek Aurora flow cytometer (Cytek, Fremont, CA, USA). Cells were gated for single, viable leukocytes. After exclusion of lymphocytes and monocytes, macrophages were identified as CD64+ CD11b+ cells. Timd4 was used as a marker for long-term residual macrophages, CCR2 as marker for monocyte derived macrophages (21 (link)).
5
Comprehensive Immune Profiling of PBMCs
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PBMCs of the patient (13-months-old) and his mother were stained with viability dye (eFluor780; cat. no. 65-0865-14; Invitrogen; Thermo Fisher Scientific, Inc.) and antibodies against CD3 (PE/Cy7; cat. no. 300420; BioLegend, Inc.), CD56 (APC; cat. no. 17-0566-42; Invitrogen; Thermo Fisher Scientific, Inc.) and CD16 (FITC; cat. no. 11-0168-42; Invitrogen; Thermo Fisher Scientific, Inc.) or antibodies against CD3 (PE/Cy7; cat. no. 300420; BioLegend, Inc.), TIGIT (PE; cat. no. 12-9500-42; Invitrogen; Thermo Fisher Scientific, Inc.) and CD279 (PD-1, FITC; cat. no. 367412) with the eBioscience intracellular fixation and permeabilization buffer set (cat. no. 88-8824-00; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instruction. Briefly, 1 million PBMCs were fixed in 100 µl IC Fixation Buffer for 30 min at RT and rinsed twice with 200 µl 1X permeabilization buffer. The cells were then resuspended in 50 µl 1X permeabilization buffer with the corresponding primary antibodies (1:100) for 30 min at RT protected from light. The stained cells were analyzed with a 5-laser Cytek Aurora flow cytometer (Cytek Biosciences). The data were analyzed with FlowJo X (v.10.0.7 R2; FlowJo LLC). The low proportion of NK cells would not alter the profile of the total CD3+ T cells.
6
Tumor Single-Cell Isolation and Analysis
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Tumors were first weighed and then minced with scissors in RPMI and filtered through a 70-μm nylon filter (BD Biosciences) in RPMI to generate single-cell suspensions. The suspensions were purified on a Ficoll gradient to eliminate dead cells and treated with red blood cell lysis buffer (ACK Lysing Buffer, Lonza) and further washed and resuspended in FACS buffer (PBS/0.5% albumin) before incubation with antibodies. The antibodies used for staining are listed in Supplemental Table 2 . In specified experiments, cell numbers were calculated per gram of tumor, and the ratio of CD8 to CD11b calculated from the absolute number of cells/gram of tumor. In experiments to detect SA-Spider-gal by flow cytometry, Spider β-Gal kits (Dojindo Laboratories) were used per the manufacturer’s protocol, prior to staining with antibodies. In experiments to study TCRs, tetramers of the following epitopes were used: hGP10025–33 (KVPRNQDWL)–Alexa Fluor 488 and OT-1 SIINFEKL-PE (NIH). Samples were acquired on a Cytek Aurora flow cytometer. Data analyses were performed using FlowJo v10 (FlowJo).
7
Multiparametric Flow Cytometry Analysis
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Cells were stained with fluorochrome-conjugated monoclonal antibodies against mouse CD8 (Cat# 100748, Biolegend, Cat# 45-0081-82, eBioscience), mouse CD4 (Cat# 100453, Biolegend, Cat# 100531, Biolegend), mouse CD45 (Cat# 103151, Biolegend, Cat# 47-0451-82, eBioscience), mouse Foxp3 (Cat# 48-5773-82, Thermo Fisher Scientific, Cat# 17-5773-82, eBioscience), human CTLA-4 (Cat# 369604, Biolegend), and Mouse IgG2a, κ Isotype Ctrl CTLA-4 (Cat# 400214, Biolegend). All the samples were stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Cat# L34957, Thermo Fisher Scientific) to exclude the dead cells. Intracellular staining was performed with the Intracellular Fixation and Permeabilization kit (Cat# 88-8824, eBioscience) according to the manufacturer’s instructions. The samples were analyzed by the BD Canton II or Cytek ™ Aurora Flow cytometer and data were analyzed by Flowjo software.
8
Assessing Cell Cycle Effects of Compounds
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To assess the effect of the compounds on cell cycle distribution, cell DNA content was assessed by flow cytometry, following a previously described protocol [44 (link)]. Briefly, A549 and H1975 cells were seeded in 6-well plates at approximately 5 × 104 and 6 × 104 cells/well, respectively, and incubated for 24 h. After this period, the culture medium was changed, and cells were exposed to MnTnHex (0.5 and 1 µM) and cisplatin (1 µM), both alone or in combination, for 72 h. The medium was collected, and the cells were detached using a 5 mM EDTA solution in PBS at 37 °C. Next, they were washed with cold PBS and fixed with ice-cold 80% ethanol. Cells were stained with propidium iodide (10 µg/mL) and treated simultaneously with RNase A (20 µg/L) for 15 min and were then analyzed using a Cytek Aurora flow cytometer (Cytek Biosciences, Fremont, CA, USA). Data acquisition was performed using Cytek SpectroFlo software (Cytek Biosciences, Fremont, CA, USA) and was analyzed with FlowJo® (Tree Star Inc., San Carlos, CA, USA). Three to four independent experiments were performed.
9
Characterizing sEV Surface Protein Markers
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To assess the expanded surface protein markers of sEVs, a MACSPlex human Exosome kit (Miltenyi Biotec, Bergisch-Gladbach, Germany) was used following the manufacturer's protocol. The sEVs were captured using 37 distinct surface marker antibodies (CD1c, CD2, CD3, CD4, CD8, CD9, CD11c, CD14, CD19, CD20, CD24, CD25, CD29, CD31, CD40, CD41b, CD42a, CD44, CD45, CD49e, CD56, CD62p, CD63, CD69, CD81, CD86, CD105, CD133.1, CD142, CD146, CD209, CD326, HLA-ABC, HLA-DR DP DQ, MCSP, ROR1, and SSEA-4) simultaneously and include the two isotype controls (mIgG1 and REA control) corresponding to the antibodies conjugated with fluorescent beads and then analyzed via flow cytometry. The samples were run on a Cytek Aurora Flow Cytometer (Cytek Biosciences, Fremont, CA, USA) and analyzed with SpectroFlo software (Cytek Biosciences).
10
Isolation and Analysis of Mouse Immune Cells
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Mouse spleens were excised and ground through a 70-µm nylon mesh. Single spleen cells were then resuspended in PBS and lysed by Red Blood Cell Lysis Buffer (cat. no. C3702; Beyotime Institute of Biotechnology). Cells were Fc-blocked with TruStain FcX™ (anti-mouse CD16/3; Clone 93; BioLegend, Inc.) for 30 min at 4°C. In addition, mouse tumor tissues were minced, underwent enzymatic digestion with 0.1% collagenase (cat. no. C5138; MilliporeSigma), hyaluronidase (0.1 mg/ml; cat. no. H6254; MilliporeSigma) and DNase IV (20 U/ml; cat. no. D5025; MilliporeSigma) in Hank's balanced salt solution (Gibco; Thermo Fisher Scientific, Inc.) and ground through a 70-µm nylon mesh. The filtrate was collected and lysed with the Red Blood Cell Lysis Buffer. A single cell suspension of 1-2×106 tumor-infiltrating lymphocytes and spleen cells was used for cell surface and intracellular marker staining. The single cell suspension was incubated with a conjugated monoclonal antibody (BioLegend, Inc., unless otherwise stated) at 4°C for 30 min. All antibodies used for flow cytometric analysis of mouse tumor samples and spleen are shown in Table SII . All samples were assessed using a Cytek Aurora flow cytometer (Cytek Biosciences) and analyzed using FlowJo software (FlowJo V10.5.3; Becton Dickinson).
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