Human colon cancer HCT 116 cells grown in adherent condition, serum starved overnight followed by treatment with vehicle or IGF1 (100 ng/ml) for 48 hours, were trypsinized and single cells were re-suspended at one million/ml in PBS buffer. Cells were incubated with conjugated antibody for 30 minutes at 4°C and washed once with PBS buffer prior to analysis. Following antibody and dilution were used: CD133/1 (AC133)-APC human (1:33 dilution) (Cat # 130-090-826, Miltenyi Biotec, Auburn, CA), CXCR4 (anti-mouse CD184)-PE conjugated clone 2B11 (Dilution 1:50) (Cat # 12-9991-82, eBioscience, San Diego, CA), and Anti-LGR5 mouse mAb, clone 2A2, PE conjugated (Dilution 1:50) (Cat # TA400001, Origene, Rockville, MA). Cell sorting was performed using FACS Aria™ II High-Speed Cell Sorter (BD Biosciences, San Jose, CA) and data were analyzed using FCS express 4 flow research edition (De-Novo Software Los Angeles, CA).
Facsaria 2 high speed cell sorter
Manufactured by BD
Sourced in United States
The FACSAria II High-Speed Cell Sorter is a flow cytometry-based instrument designed for the rapid and precise sorting of individual cells from complex samples. It is capable of analyzing and sorting multiple cell populations simultaneously, providing researchers with a powerful tool for applications such as cell line development, cell-based assays, and sample preparation for downstream analysis.
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Lab products found in correlation
Fcs express 4 flow cytometry software, by De Novo Software (3 mentions)
Facsdiva software, by BD (3 mentions)
Lgr5 pe, by OriGene (2 mentions)
Red blood cell lysis buffer, by BD (2 mentions)
Fcs express 4 flow research edition, by De Novo Software (1 mentions)
Anti fitc magnetic microbeads, by Miltenyi Biotec (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Liberase, by Roche (1 mentions)
Cd11c microbeads, by Miltenyi Biotec (1 mentions)
Alexa fluor 647 mouse anti stat3 py705, by BD (1 mentions)
Canto 2, by BD (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Verapamil, by Merck Group (1 mentions)
Dclk1, by Abcam (1 mentions)
30 μm cell strainer, by Miltenyi Biotec (1 mentions)
Female balb c mice, by Charles River Laboratories (1 mentions)
Streptomycin, by Corning (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
13 protocols using facsaria 2 high speed cell sorter
1
Profiling Colon Cancer Stem Cell Markers
2
Isolation and Sorting of Dendritic Cell Subsets
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For ex vivo DC isolation, the thymuses or spleens were pooled and cut with blunt scissors before digestion in Roswell Park Memorial Institute (RPMI) 1640 with 2% fetal bovine serum (FCS) supplemented with Liberase (200 μg/ml, Roche) and DNase I (40 μg/ml, Sigma-Aldrich) for 10–15 min. After incubation with 2.4G2 antibody, DCs were isolated using CD11c MicroBeads (Miltenyi Biotec), according to manufacturer's instruction. Each of the cDC subsets and pDCs was subsequently examined by fluorescence-activated cell sorting using a FACSAria™ II high-speed cell sorter (BD Biosciences) following staining with F4-80-, CD45.1- or CD45.2-, CD11c-, CD11b-, B220-, CD24-, and Sirpα-specific antibodies.
For pre-cDC and pDC isolation, BM was extracted from the femurs and tibias, and erythrocytes removed using Gey's treatment. Lineage cells were first removed using fluorescein isothiocyanate (FITC) conjugate anti-CD3, anti-CD19, anti-NKP46, and, for pre-cDC isolation only, anti-B220, and anti-FITC magnetic MicroBeads (Miltenyi Biotec). Pre-cDCs and pDCs were subsequently FACS-sorted following staining with anti-MHC II-, CD135-, Sirpα- and CD11c-specific antibodies or CD19-, B220-, and CD11c-specific antibodies, respectively.
For pre-cDC and pDC isolation, BM was extracted from the femurs and tibias, and erythrocytes removed using Gey's treatment. Lineage cells were first removed using fluorescein isothiocyanate (FITC) conjugate anti-CD3, anti-CD19, anti-NKP46, and, for pre-cDC isolation only, anti-B220, and anti-FITC magnetic MicroBeads (Miltenyi Biotec). Pre-cDCs and pDCs were subsequently FACS-sorted following staining with anti-MHC II-, CD135-, Sirpα- and CD11c-specific antibodies or CD19-, B220-, and CD11c-specific antibodies, respectively.
3
Immunophenotyping of Cells by Flow Cytometry
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Cells were collected with trypsinization and stained with indicated antibody. For intracellular stains, cells were fixed (BD catalogue # 558049), and permeabilized (BD catalogue # 558050) prior to analysis. Alexa Fluor 647 Mouse Anti-Stat3 (pY705) (Cat# 557815, BD) and Alexa Fluor® 647 Mouse IgG2a, Isotype Control (Cat# 558053, BD) antibodies were used and flow cytometry was performed on BD canto II. The data were analyzed using FlowJO (Tree Star Inc.) software. For surface stains, cells were incubated with human CD133-APC (Cat# FAB11331A-100, R&D systems) conjugated antibody for 30 min at 4 °C and washed once with PBS buffer prior to analysis. Cell sorting was performed using FACSAria II High-Speed Cell Sorter (BD Biosciences, San Jose, CA) and data were analyzed with FCS Express 4 Flow Cytometry software (De-Novo Software, Los Angeles, CA).
4
Isolation and Analysis of Cancer Stem-like Cells
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The TSCC side population was sorted as previously described 29 (link). Briefly, TSCC cells were incubated at 37 °C for 90 minutes in the dark with 5 μg/ml Hoechst 33342 (Sigma, San Louis, MO, USA) and intermittent mixing either alone or in the presence of 50 μM verapamil (Sigma, San Louis, MO, USA), an inhibitor of ABC transporters. The cells were then centrifuged and resuspended in ice-cold HBSS. The samples were sorted using a FACSAria II high-speed cell sorter (BD Biosciences, San Diego, CA, USA). Propidium iodide (2 μg/ml) was added to exclude dead cells. Hoechst 33342 was excited with the UV laser at 350 nm, and the fluorescence emission was measured with 405 / BP30 (Hoechst blue) and 570 / BP20 (Hoechst red) optical filters. After sorting, the SP and non-SP cells were used for further experiments.
5
Analyzing Stem Cell Markers in Colon Cancer
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Human colon
cancer HT-29 cells,
grown in spheroid or monolayer condition were treated with vehicle
or NSGMs for 24 h, were trypsinized and single cells were resuspended
at 106 cells/mL in PBS buffer. Cells were incubated with
fluorophore conjugated antibody for 30 min at 4 °C and washed
once with PBS buffer prior to analysis. Following antibody and dilution
were used: LGR5-PE) (Dilution 1:50, Origene), DCLK1(Dilution 1:33,
Abcam). Cell sorting was performed using FACSAria II High-Speed Cell
Sorter (BD Biosciences) and data were analyzed with FCS Express 4
Flow Cytometry software (De-Novo Software).
cancer HT-29 cells,
grown in spheroid or monolayer condition were treated with vehicle
or NSGMs for 24 h, were trypsinized and single cells were resuspended
at 106 cells/mL in PBS buffer. Cells were incubated with
fluorophore conjugated antibody for 30 min at 4 °C and washed
once with PBS buffer prior to analysis. Following antibody and dilution
were used: LGR5-PE) (Dilution 1:50, Origene), DCLK1(Dilution 1:33,
Abcam). Cell sorting was performed using FACSAria II High-Speed Cell
Sorter (BD Biosciences) and data were analyzed with FCS Express 4
Flow Cytometry software (De-Novo Software).
6
Isolation and Culture of Murine Bone Marrow-Derived Macrophages
7
Isolating Leukemic and Normal Mouse Cells
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Femoral bone marrow was collected with cold PBS, supplemented with 1% fetal bovine serum (FBS), and subjected to red blood cell lysis buffer (BD Biosciences, #555899). Given that the mice had been injected with human pre-B acute lymphoblastic leukemia (ALL) cells at one day prior to initiation of the chronic restraint stress period, fluorescent activated cell sorting was used to separate ALL cells from normal mouse leukocytes at the conclusion of the experiment. To mark ALL cells, bone marrow cell samples were incubated with PE-conjugated antibodies against human CD10 (BD Biosciences, #555375), washed, and sorted on a FACSAria II High-Speed Cell Sorter with FACSDiva software (BD Biosciences). Sorted human-CD10− mouse leukocytes were stored at −80 °C until processing for total RNA extraction. To control for potential effects of leukemia burden on biological aging in normal mouse leukocytes, the amount of ALL tumor load, as measured in Lamkin et al. (2012) (link), was used as a covariate in the present analyses.
8
LGR5 Expression in Colon Cancer Spheroids
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Human colon cancer HT29 cells, grown in spheroid condition and treated with vehicle or GAG for 24 hrs, were trypsinized and single cells were re-suspended at 106 cells/mL in PBS buffer. Cells were incubated with LGR5-PE (anti-human mouse monoclonal MAB clone 2A2) (Dilution 1:50) (Origene, Rockville, MA) antibody for 30 min at 4 ºC and washed once with PBS buffer prior to analysis. Cell sorting was performed using FACSAria™ II High-Speed Cell Sorter (BD Biosciences, San Jose, CA) and data were analyzed with FCS Express 4 Flow Cytometry software (De-Novo Software, Los Angeles, CA).
9
FACS-based Screening of Bacterial Mutants
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Cells grown in 5 ml 2xTY medium were used to inoculate a 50 ml preculture in CGXII minimal medium with 100 mM glucose and cultivated for approx. 16 h at 130 rpm and 30°C.
The cells of this preculture were washed once in 0.9% (wt/vol) NaCl solution and then used to inoculate the main cultures in 500 ml baffled shake flasks containing 50 ml CGXII medium with 100 mM glucose to an OD 600 of 1. The main cultures were incubated at 130 rpm and 30°C until the exponential growth phase was reached (approx. 4-5 h). Culture samples were removed from the flasks and diluted in FACS flow buffer (Agilent) to an OD 600 below 0.1 prior to FACS analysis using an FACS ARIA II high-speed cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) as described previously (Kortmann et al., 2015) . Mutant cells from 1:1 mixtures with wild-type cells were sorted on BHI agar plates using the gates P2 and P3 as indicated in Fig. 5 and Fig. 6. The BHI plates were incubated at 30°C for at least 24 h before the colonies were tested for deletion of cyaB and cpdA by colony PCR using the oligonucleotide pairs cyaB_deltest_fw/cyaB_deltest_rv and cpdA_deltest_fw/cpdA_deltest_rv.
The cells of this preculture were washed once in 0.9% (wt/vol) NaCl solution and then used to inoculate the main cultures in 500 ml baffled shake flasks containing 50 ml CGXII medium with 100 mM glucose to an OD 600 of 1. The main cultures were incubated at 130 rpm and 30°C until the exponential growth phase was reached (approx. 4-5 h). Culture samples were removed from the flasks and diluted in FACS flow buffer (Agilent) to an OD 600 below 0.1 prior to FACS analysis using an FACS ARIA II high-speed cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) as described previously (Kortmann et al., 2015) . Mutant cells from 1:1 mixtures with wild-type cells were sorted on BHI agar plates using the gates P2 and P3 as indicated in Fig. 5 and Fig. 6. The BHI plates were incubated at 30°C for at least 24 h before the colonies were tested for deletion of cyaB and cpdA by colony PCR using the oligonucleotide pairs cyaB_deltest_fw/cyaB_deltest_rv and cpdA_deltest_fw/cpdA_deltest_rv.
10
Phenotyping T Cell Subsets
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Cells isolated from DLNs and expanded as described above were stained with a panel of antibodies and analyzed or sorted based on surface marker expression on a BD FACS Aria™ II High-Speed Cell Sorter at days 0 (after B/I), 1, 3 and 6 of expansion. Fluorescent labeled Abs directed against the following markers were obtained from Biolegend: CD4 (GK1.5), CD8 (53–6.7), CD44 (IM7), CD62L (MEL-14). Appropriate isotype and single color controls were used in all cases. T cell subsets analyzed were T effector (TE) CD44+CD62L-, T effector memory (TEM) CD44+CD62Llow, and T central memory (TCM) CD44+CD62Lhigh.
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