To analyze cell cycle distribution, IMR-32 and SH-SY5Y cells (1×106) were quickly fixed by methanol (4°C) and treated with 50 µg/ml propidium iodide (PI) (Thermo Fisher Scientific, USA) for 20 min. A FACSCalibur™ flow cytometer (Thermo Fisher Scientific) was used to obtain the cell cycle histograms. MultiCycle AV software (Phoenix Flow System, USA) was then used to calculate the percentages of cells in the G0/G1, S or G2/M phases.
Facscalibur flow cytometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FACSCalibur flow cytometer is a versatile and reliable instrument designed for multicolor flow cytometric analysis. It features advanced optics and detection capabilities, enabling the simultaneous measurement of multiple parameters on individual cells or particles in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (3 mentions)
Fxcycle pi rnase staining solution, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Cytofix cytoperm buffer, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Sodium chlorate, by Merck Group (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Mouse anti cd3 cd28 antibodies, by Thermo Fisher Scientific (1 mentions)
Anti mouse ifn γ, by Thermo Fisher Scientific (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Anti cd3 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd4 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd19 apc, by Thermo Fisher Scientific (1 mentions)
Anti igm fitc, by BioLegend (1 mentions)
Anti cd8 apc, by BioLegend (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
19 protocols using facscalibur flow cytometer
1
Cell Cycle Analysis by Flow Cytometry
2
Heparinase-Mediated Cell Surface Glycosaminoglycan Analysis
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One million cells were treated with 2 U of a heparinase I and III blend from Flavobacterium heparinum (no. H3917; Merck) for 1 h at 37°C and 5% CO2 in DMEM, and then cells were reverse transfected with 2 μg of GFP using Lipofectamine 2000 (Invitrogen) in 6-well plate.
HCT116cas9 cells were grown in 50 mM sodium chlorate (no. 1.06420; Merck), DMEM, and 10% FBS for at least 48 h, and then 150,000 cells were reverse transfected with 500 ng of GFP using Lipofectamine 2000 (Invitrogen) in a 24-well plate.
At 24 (heparinase) or 48 (sodium chlorate) hours posttreatment, cells were detached using PBS and 0.02% EDTA, and then heparan sulfate was stained using 1:30 of F58-10E4 as the primary antibody (catalog no. 370255-S; Amsbio) in PBS and 3% bovine serum albumin (BSA) for 30 to 40 minutes on ice. Next, cells were washed with PBS and 1% FBS, incubated with 1:30 anti-mouse Alexa Fluor 594 (A-11032; Thermo) in PBS and 3% BSA, washed twice using PBS and 1% FBS, and then analyzed on a FACSCalibur flow cytometer.
HCT116cas9 cells were grown in 50 mM sodium chlorate (no. 1.06420; Merck), DMEM, and 10% FBS for at least 48 h, and then 150,000 cells were reverse transfected with 500 ng of GFP using Lipofectamine 2000 (Invitrogen) in a 24-well plate.
At 24 (heparinase) or 48 (sodium chlorate) hours posttreatment, cells were detached using PBS and 0.02% EDTA, and then heparan sulfate was stained using 1:30 of F58-10E4 as the primary antibody (catalog no. 370255-S; Amsbio) in PBS and 3% bovine serum albumin (BSA) for 30 to 40 minutes on ice. Next, cells were washed with PBS and 1% FBS, incubated with 1:30 anti-mouse Alexa Fluor 594 (A-11032; Thermo) in PBS and 3% BSA, washed twice using PBS and 1% FBS, and then analyzed on a FACSCalibur flow cytometer.
3
Annexin V-FITC/PI Apoptosis Assay
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Annexin 8-fluorescein isothiocyanate (FITC)/propidium iodide (PI) method was adopted to detect cell apoptosis. Cells were incubated 6-well plates for 48 h, washed with 1× binding buffer (185 mL), and incubated with 5 µL AnnexinV FITC and 5 µL PI. Cell apoptosis was detected by FACSCalibur flow cytometer and Cell Quest Pro software (Thermo Fisher). The data were analyzed by FlowJo 7.6 software [23 (link)].
4
Heparinase and Sodium Chlorate Treatment
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Heparinase: 1.10 6 were treated with 2U of Heparinase I and III blend from Flavobacterium heparinum (Merck -H3917) for 1 h at 37°C 5% CO2 in DMEM and then cells were reverse transfected with 2 µg of GFP using lipofectamine2000 (Invitrogen) in 6 well plate.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted October 14, 2020. ; https://doi.org/10.1101/2020.05.20.105528 doi: bioRxiv preprint 20 Sodium chlorate: HCT116cas9 cells grow in 50 mM sodium chlorate (Merck -1.06420) DMEM 10 % FBS for at least 48 h then 150 000 cells were reverse transfected with 500 ng of GFP using lipofectamine 2000 (Invitrogen) in 24 well plate. 24 (heparinase) or 48 (sodium chlorate) hours post treatment, cells were detach using PBS 0.02% EDTA, then heparan sulfate was stained using 1:30 of F58-10E4 as primary antibody (AMSBIO, cat#370255-S) in PBS 3% BSA for 30 to 40 minutes on ice then washed with PBS 1% FBS and incubated with 1:30 anti-mouse Alexa Fluor 594 (Thermo, A-11032) in PBS 3% BSA, washed twice using PBS 1% FBS then and analysed on a FACSCalibur flow cytometer.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted October 14, 2020. ; https://doi.org/10.1101/2020.05.20.105528 doi: bioRxiv preprint 20 Sodium chlorate: HCT116cas9 cells grow in 50 mM sodium chlorate (Merck -1.06420) DMEM 10 % FBS for at least 48 h then 150 000 cells were reverse transfected with 500 ng of GFP using lipofectamine 2000 (Invitrogen) in 24 well plate. 24 (heparinase) or 48 (sodium chlorate) hours post treatment, cells were detach using PBS 0.02% EDTA, then heparan sulfate was stained using 1:30 of F58-10E4 as primary antibody (AMSBIO, cat#370255-S) in PBS 3% BSA for 30 to 40 minutes on ice then washed with PBS 1% FBS and incubated with 1:30 anti-mouse Alexa Fluor 594 (Thermo, A-11032) in PBS 3% BSA, washed twice using PBS 1% FBS then and analysed on a FACSCalibur flow cytometer.
5
Multiplex Cytometric Assay for Th-Related Cytokines
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Serum levels of Th-related cytokines that included Th1
(IFN-γ, TNF-α and IL-2), Th2 (IL-4, IL-5, IL-6, IL-10 and IL-
13 ), Th17 (IL-17A, IL-17F and IL-21), Th9 (IL-9) and Th22
(IL-22) were quantified with a multiplex cytometric bead
assay using a commercial kit (BioLegend, San Diego, CA,
USA) according to the manufacturer’s directions. Briefly, a
mixture of FITC-labeled antibody-coated beads for each
desired cytokine, which could be differentiated by their sizes
and fluorescence intensities, was incubated with the mouse
serum samples or standards. After capturing the cytokines by
the beads, biotin-conjugated anti-mouse antibody and PElabeled streptavidin were successively added. The results were
visualized with a FACSCalibur flow cytometer (eBioscience,
San Diego, CA, USA) and the data were analyzed with
FlowCytomix Pro-3.0 software (BioLegend).
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Serum levels of Th-related cytokines that included Th1
(IFN-γ, TNF-α and IL-2), Th2 (IL-4, IL-5, IL-6, IL-10 and IL-
13 ), Th17 (IL-17A, IL-17F and IL-21), Th9 (IL-9) and Th22
(IL-22) were quantified with a multiplex cytometric bead
assay using a commercial kit (BioLegend, San Diego, CA,
USA) according to the manufacturer’s directions. Briefly, a
mixture of FITC-labeled antibody-coated beads for each
desired cytokine, which could be differentiated by their sizes
and fluorescence intensities, was incubated with the mouse
serum samples or standards. After capturing the cytokines by
the beads, biotin-conjugated anti-mouse antibody and PElabeled streptavidin were successively added. The results were
visualized with a FACSCalibur flow cytometer (eBioscience,
San Diego, CA, USA) and the data were analyzed with
FlowCytomix Pro-3.0 software (BioLegend).
6
Immunosuppressive Capacity of Irradiated MSCs
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Murine MSCs with different treatments were irradiated with 30 Gy from a 137 Cs source as previously described11 (link), to inactivate MSCs by inhibiting their proliferation while reserving their immunosuppressive capacity, and then seeded into 96-well plates. Freshly isolated splenocytes (2 × 105 cells/well) from C57/BL6 mice were labeled with 7.5 μM carboxyfluorescein diacetatesuccinimidyl ester (CFSE, Thermo Fisher Scientific) and co-cultured with murine MSCs for 3 days in the presence of mouse anti-CD3/CD28 antibodies (eBiosciences, San Diego, CA, USA), then collected for flow cytometric analysis on a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Human MSCs with different treatments were irradiated with 30 Gy from a 137 Cs source and seeded into 96-well plates. Freshly isolated PBMCs (2 × 105 cells/well) from healthy volunteers were labeled with 7.5 μM CFSE and co-cultured with human MSCs for 3 days in the presence of human anti-CD3/CD28 antibodies (eBiosciences), then collected for flow cytometric analysis on a FACS Calibur flow cytometer.
7
Intracellular Cytokine Staining of T Cells
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For intracellular staining, T cells (2 × 105 in 100 μl of PBS) harvested 5 days post in vitro stimulation with the immunizing peptide and APCs were incubated for 4 h with 50 ng/mL of PMA, 1 μg/mL of ionomycin, and 1 μg/mL of brefeldin A (Sigma-Aldrich), then were washed, fixed, permeabilized overnight with Cytofix/Cytoperm buffer (eBioscience), intracellularly stained with antibodies against IFN-γ and IL-17, and analyzed on a FACScalibur flow cytometer.
8
Intracellular Cytokine Staining of T Cells
9
Cytokine Expression in Colitis Mice
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For staining for IL-17RA, CECs were collected from TNBS-induced colitis mice or control mice, and then were stained with phycoerythrin (PE)-conjugated anti-mouse IL-17RA antibodies (Biolegends). For staining IFN-r within CD4+T cells and IL-12 within monocytes/macrophage, cells were stimulated for 4 h with 50 ng/ml of phorbol 12-myristate 13-acetate, 1 µg/ml of ionomycin, and 1 µg/ml of brefeldin A (Sigma, St Louis, MO), then were washed and stained with fluorescein isothiocyanate (FITC)-conjugated anti-human CD4, anti-mouse CD4, anti-human CD14 or anti-mouse CD11b, then fixed for overnight with Fix/Perm buffer, washed with permeabilization buffer, stained for 30 min at 4°C with PE-conjugated anti-human IFN-γ, anti-mouse IFN-γ, anti-human IL-12P70 and anti-mouse IL-P70 antibodies(all from eBioscience) and analyzed on a FACScalibur flow cytometer.
10
Quantifying Apoptosis via Flow Cytometry
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An apoptosis assay was performed on mock- or NS1-transfected cells using the PE Annexin V apoptosis detection kit according to the manufacturer's instructions (BD PharMingen, San Diego, CA, Catalogue number: 559763). PE Annexin V was used to quantitatively determine the percentage of cells undergoing the early phases of apoptosis through bounding to phospholipid phosphatidylserine. In this kit, 7-AAD is also used as a vital dye to distinguish viable from nonviable cells. Cells were analyzed using a FACSCalibur flow cytometer (eBioscience, Santa Cruz, CA, USA). Cells that stained double-positive for PE Annexin V and 7-AAD were considered as apoptotic cells.
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