Cytokine production was assessed for specific T-cell subsets at the single-cell level using intracellular flow cytometry. PBMCs (1×106) were cultured in 1.0 ml culture media containing 10 ng/ml PMA, 1.0 μg/ml ionomycin and 3 μM monensin (Sigma-Aldrich). Cultures were incubated overnight (6 h) at 4 °C. Following incubation, the supernatants were removed and the cells were fixed in 4.0% paraformaldehyde for 10 min. To detect intracellular production of IFNγ or IL-2 (following surface marker staining), the fixed PBMCs were resuspended in 200 μl of permeabilization buffer (5.0% nonfat dry milk and 0.5% saponin in phosphate-buffered saline (PBS)) to which 0.5 μg of labeled mouse antibody to human IL-2 (FITC), IFNγ (PE), CD8 (ECD), and CD3 (PC5) was added. Fluorescent-labeled antibodies were obtained from Beckman-Coulter (Miami, FL, USA). The cells were incubated at 4°C for 16 h and then washed in PBS-containing saponin. The cells were then resuspended in 1.0% paraformaldehyde and analyzed on a Beckman Coulter XL flow cytometer. The gating strategy consisted of T-cell identification, resolution of CD4 and CD8 subsets, followed by resolution and enumeration of cells producing IFNγ and IL-2.
Xl flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The XL flow cytometer is a laboratory equipment designed for analyzing and sorting cells or particles in a fluid sample. It utilizes the principles of hydrodynamic focusing and fluorescence detection to provide rapid and precise measurements of various cellular properties, such as size, granularity, and the presence of specific markers on the cell surface or within the cell.
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Lab products found in correlation
Modfit cell cycle analysis software, by Verity Software House (2 mentions)
Cell quest, by Verity Software House (2 mentions)
Monensin, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Mpl cellmek, by Beckman Coulter (1 mentions)
Facscount system, by BD (1 mentions)
Flow count fluorosphere, by Beckman Coulter (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Keygen Biotech (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
7 protocols using xl flow cytometer
1
Single-Cell Cytokine Profiling of T Cells
2
Cell Cycle Analysis of Prostate Cancer Cells
3
Evaluation of FACSPresto CD4 Enumeration
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Phase I was done under ideal laboratory conditions to assess the baseline accuracy and precision of the instrument, compared to the predicate method of CD4 testing in the NHLS. The latter, i.e. FlowCare™ Panleuco-gated (PLG/CD4) 2-color (CD45 FITC/CD4-RD1) single platform cell enumeration (with addition of Flow Count fluorospheres, Beckman Coulter) [36 (link)–42 (link)]. Testing was done on a Beckman Coulter MPL/CellMek fully automated flow cytometer system using NHLS standardized standard operating procedures (SOPs). Remnant K3EDTA blood was obtained from the CD4 testing laboratory within 24 hours of predicate CD4 testing, and used to manually fill FACSPresto™ cartridges by pipetting EDTA blood into cartridges (n = 214). Up to ten patient samples (cartridges) were set up simultaneously and incubated at room temperature for 18 minutes before analysis on the instrument, with individual results printed within 2–3 minutes. A subset (n = 115/214) of samples were also tested on a 2nd BD platform (industry standard for small to medium volume CD4 testing), the BD FACSCount™ system [43 (link)–45 (link)], and the reference Tetrachrome™ (CD45FITC/CD4-RD1/CD8-ECD/CD3-PC5) T-cell enumeration method on the Beckman Coulter XL flow cytometer [46 (link)].
4
Cell Cycle Analysis of Prostate Cancer Cells
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LNCaP and DU145 cells, 70% confluent, were starved for 36 h in 1% FBS to arrest them in G1 phase of the cell cycle, after which they were treated with 20 μM BA in RPMI 1640 complete media for 48 h. Following treatment cells were collected, washed twice with chilled phosphate-buffered saline (PBS) and spun in a cold centrifuge at 600× g for 10 min. The pellet was fixed and resuspended in 50 μL PBS and 450 μL chilled methanol for 1 h at 4 °C. The cells were washed twice with PBS at 600× g for 5 min and again suspended in 500 μL PBS and incubated with 5 mL RNase (20 μg/mL final concentration) for 30 min at 37 °C. The cells were chilled over ice for 10 min and stained with propidium iodide (50 μg/mL final concentration) for 1 h. They were analyzed by Beckman Coulter XL flow cytometer (Indianapolis, IN, USA) and evaluated using Cell Quest and ModFit cell cycle analysis software (Verity Software House, Topsham, ME, USA).
5
MSC Phenotyping by Flow Cytometry
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MSCs phenotype was confirmed by flow cytometry based on the positivity for CD29, CD44, Sca-1, and CD90 and the absence of CD45 and CD34 antigens (all antibodies were purchased from BD Biosciences, conjugated to FITC or PE). Surface staining was performed following standard protocol. The samples were acquired in a Beckman Coulter XL flow cytometer. Data were analyzed using FCS Express 4 Plus Research Edition software.
6
Apoptosis and Viability Assays
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The apoptosis assays were performed using flow cytometry with an Annexin V-FITC/PI apoptosis detection kit (KeyGEN). After centrifugation, cells were resuspended in 500 µl binding buffer containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI), and incubated in the dark for 15 min. The Beckman-Coulter XL flow cytometer was used for the measurements with the emission at 530/630 nm. For the cell viability assays, the Cell Counting Kit-8 (CCK-8; #C0037, Beyotime) was added into the MLO-Y4 cells, and then incubated at 37 °C for 2 h. The absorbance was determined using a microplate reader (Bio-Rad) at 450 nm.
7
Phagocytosis Assay of Bovine PMNL
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Phagocytosis was performed according to procedures described by Garcia et al. (2015) . Briefly, fluorescent beads (Fluoresbrite Yellow-Green Carboxylate 1.75μm Microspheres, PolySciences, Warrington, PA) were precoated with heat-inactivated FBS (250 μL of FBS per 1 μL of beads; Sigma-Aldrich Co.) at room temperature for 45 min. To each cell population, 100 μL of the FBS-coated beads was added to obtain a 10:1 ratio (beads:PMNL). Samples were run in duplicate and incubated for 2 h at 37°C, 5% CO 2 , and 95% relative humidity, and then centrifuged for 5 min (1,000 × g at 4°C). The pellets were washed in 1 mL of CMF-HBSS and fixed with 300 μL of 4% paraformaldehyde (Sigma-Aldrich Co.). The PMNL population was gated by forward and side scatters (XL Flow Cytometer, Beckman Coulter, Fullerton, CA). Data were collected on 20,000 events per sample in the gate set for bovine PMNL, and percentage fluorescence was used as the quantitative index of PMNL response. Phagocytosis by PMNL was expressed as the total percentage of PMNL engulfing one or more beads.
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