Mice were sacrificed by CO2 inhalation, and whole blood was immediately collected in EDTA-coated tubes. Red blood cells in whole blood were lysed with lysis buffer prior staining. Tibias and femurs were surgically removed and bone marrow cells flushed with collection media (DMEM supplemented with 2% FBS, 50 IU/ml penicilin, 50ug/ml streptomycin.) Spleens were minced in collection media and filtered through cell strainers. Single cell suspensions were stained with fluorescently labeled antibodies and DAPI. The following antibodies were used: PerCpCy5.5-anti-B220 (RA3-6B2),PE-Cy7-anti-IgM (II/41),biotin-anti-IgD (11–26), APC-anti-CD93 (AA4.1) (all eBioscience), PE-anti-IgD (11-26c.2a), Alexa Fluor 700-anti-CD45.2 (104), Pacific Blue- anti-CD45.1 (A20) (all Biolegend), biotin-anti-CXCR4, PE-anti-CD19 (1D3) (all BD Biosciences) and, Qdot 605-streptavidin (Invitrogen). Flow cytometry analyses were performed on a LSR II (BD Biosciences) or FACSCalibur and data analyzed by FlowJo version 9.2 (Tree Star, Inc).
Facscalibur
Manufactured by Tree Star
Sourced in United States
The FACSCalibur is a flow cytometry instrument designed for biological research. It utilizes laser excitation and electronic detection to analyze and sort cells or particles suspended in a fluid stream. The FACSCalibur provides multiparameter analysis of cell characteristics, including size, granularity, and fluorescence intensity.
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Lab products found in correlation
Ps 1145, by Merck Group (2 mentions)
Rhodamine red x conjugated goat anti human igm chain specific fab fragment, by Jackson ImmunoResearch (2 mentions)
Alexafluor 488 conjugated goat anti human igg h l specific fab fragment, by Jackson ImmunoResearch (2 mentions)
Perm wash buffer, by BD (2 mentions)
7 aad, by BD (2 mentions)
Biotin anti cxcr4, by BD (1 mentions)
Pe anti cd19 1d3, by BD (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Anti cd4 percp, by BioLegend (1 mentions)
Il 21r fc chimera, by R&D Systems (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Rnase a, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
I ab af6 1201, by BD (1 mentions)
Facs canto, by Tree Star (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Caspase 3 activity assay kit, by Enzo Life Sciences (1 mentions)
Yo pro 1, by Thermo Fisher Scientific (1 mentions)
Infinite m200, by Tecan (1 mentions)
Fluorescent antibody, by Thermo Fisher Scientific (1 mentions)
49 protocols using facscalibur
1
Murine Hematopoietic Cell Isolation
2
Ezrin Manipulation Regulates DLBCL Viability
3
Multiparameter Flow Cytometry Analysis
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To block nonspecific binding to Fc receptors, cells were incubated with anti‐CD16/32 antibody (BD Franklin Lakes), and then stained with different panels of fluorochrome conjugated monoclonal antibodies (Supplementary table 1 ) in PBS/2% FBS. For intracellular staining of transcription factors, cells were fixed, permeabilized and stained using the transcription factor buffer set (BD Franklin Lakes) according to the manufacturer's instructions. To track divisions, CellTrace Violet (Invitrogen) was used to label cells according to the manufacturer's protocol. Samples were run using a BD LSR II, BD Fortessa, BD Verse or FACSCalibur machine and data were analyzed by FlowJo software v9.6.4 (Tree Star, Ashland, OR). For follicular B cell sorts, splenocytes were stained with LIVE/DEAD Aqua dye, B220, CD21/35 and CD23 antibodies and sorted on the FACSAria II cell sorter (BD Franklin Lakes).
4
Apoptosis induction by 4-HPPP in H1299 cells
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To examine the apoptosis-inducing potential of 4-HPPP in H1299 cells, annexin V/PI double staining was performed to detect the externalization of phosphatidylserine (PS). In brief, 2 × 105 cells were seeded onto 12-well plates and treated with or without 4-HPPP for 48 h and 72 h. Subsequently, the cells were harvested and stained with the annexin V/PI kit (Strong Biotech) according to the manufacturer's instructions. Cells were analyzed by flow cytometry (FACSCalibur) using FlowJo v7.5.5 software (Tree Star, Inc.).
5
Intracellular Cytokine Staining for IL-21
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Intracellular cytokine staining for IL-21 was performed as described previously (Suto et al., 2008 (link); Hiramatsu et al., 2010 (link)). In brief, cultured cells were washed and stimulated with PMA plus ionomycin for 5 h. Cells were stained with anti-CD4 PerCP or anti-Thy1.1 PerCP (BioLegend) for 30 min at 4°C. Cells were then fixed, permeabilized with Perm/Wash buffer (BD), and incubated with IL-21R/Fc chimera (R&D Systems) and anti–IL-4 PE, anti–IFN-γ PE, or anti–IL-17A PE for 30 min at 4°C. Cells were then washed with Perm/Wash buffer and stained with allophycocyanin-conjugated affinity-purified F(ab′)2 fragment of donkey anti–human IgG (H+L; Jackson ImmunoResearch Laboratories) for 30 min at 4°C. Cytokine profiles of CD4+ cells were analyzed on a FACSCalibur with FlowJo software (Tree Star).
6
Cell Cycle Synchronization and Apoptosis Analysis
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Cell cycle synchronization was achieved by serum starvation and thymidine double blocking (22 (link)). Subsequently, the cells were harvested and stained with 5 µl propidium iodide at 4°C for 30 min in the dark according to the manufacturer's protocol (BestBio). Flow cytometry was performed using a FACScan flow cytometer (BD Biosciences).
Apoptosis was detected using an Annexin V-APC and 7-AAD kit (BioGems International, Inc.) according to the manufacturer's protocol, and flow cytometry was performed on the cells. MNNG/HOS cells were harvested 48 h after transfection or treated with OTSSP167 (10 nM) and subsequently harvested using EDTA-free trypsin, centrifuged at 800 × g for 5 min; and, after washing twice with cold PBS, the cells were resuspended at a concentration of 1×106/ml and stained with 5 µl Annexin V-APC and 5 µl 7-AAD at room temperature for 15 min in the dark. The cells were analyzed by flow cytometry (FACSCalibur) and the results were analyzed using FlowJo software (version 10; TreeStar, Inc.).
Apoptosis was detected using an Annexin V-APC and 7-AAD kit (BioGems International, Inc.) according to the manufacturer's protocol, and flow cytometry was performed on the cells. MNNG/HOS cells were harvested 48 h after transfection or treated with OTSSP167 (10 nM) and subsequently harvested using EDTA-free trypsin, centrifuged at 800 × g for 5 min; and, after washing twice with cold PBS, the cells were resuspended at a concentration of 1×106/ml and stained with 5 µl Annexin V-APC and 5 µl 7-AAD at room temperature for 15 min in the dark. The cells were analyzed by flow cytometry (FACSCalibur) and the results were analyzed using FlowJo software (version 10; TreeStar, Inc.).
7
Elucidating BASP1's Impact on Thyroid Cancer Cell Cycle
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To determine the effect of BASP1 on the cell cycle of anaplastic thyroid cancer, BHT-101 and KMH-2 cells were seeded in a 6-well plate overnight, then pcDNA-BASP1 was added and incubated with cells for 48 h. After washed twice by PBS, the cells were fixed in 70% precooling ethanol overnight. The cells were then treated with RNaseA (50 μg/ml) and stained with propidium iodide (PI) (Sigma-Aldrich Co.) as per manufacturer's instructions. Samples were run on an FACSCalibur and analyzed their DNA content using Flowjo software (Tree Star Inc., Ashland, OR, USA).
8
Comprehensive Phenotypic Profiling of Immune Cells
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Phenotypic analysis was performed using 100 μl peripheral blood samples. Cells were stained with fluorochrome-conjugated anti-human CD3 (UCHT1), CD19 (HIB19), CD25 (BC96), CD45RA (HI100), CD45RO (UCHL1), CD62L (DREG-56), CD23 (EBVCS-5), CD154 (24-31), CCR7 (G043H7), ICOS (C398.4A), IgD (IA6-2), TCRγδ (B1) (all from Biolegend, San Diego, CA, USA) and anti-human CD27 (M-T271), CD40 (5C3), CD69 (FN50), CD80 (L307.4), CD86 (FUN-1), CXCR5 (RF8B2), HLA-DR (G46-6) (all from BD Biosciences, San Diego, CA, USA). Data were collected by flow cytometry on a FACScalibur and were analyzed with FlowJo software (TreeStar).
9
Multicolor Flow Cytometry Immunophenotyping
10
Caspase-3 Activity and Cell Apoptosis Analysis
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Caspase-3 activity was determined using a caspase-3 activity assay kit (Enzo Life Sciences, Lörrach, Germany, Cat.No. ALX-260-031). Following the manufacturer’s protocol, cell lysates were incubated with reaction buffer and caspase-3 substrate Ac-DEVD-AMC for 10 min at 37 °C. Fluorescence was measured using a TECAN Infinite M200 microplate reader with excitation and emission wavelengths of 360 and 440 nm, respectively. FACS analysis using PI and YO-PRO®-1 (Invitrogen, Cat.No. V13243) was performed on cells plated in 6-well plates at 80,000 cells/well according to manufacturer’s instructions. Fluorescence intensity was measured by flow cytometry (FACScalibur, Fortessa) and evaluated with FlowJo (Tree Star, Inc.). The data were recorded for a total of 15,000 events per sample.
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