MDA-MB-231 or 4T1 cells were injected i.v. into female CD1 nude or balb/c mice (4–6 weeks) respectively. Animals were sacrificed at defined time points and lungs were harvested. Lung metastasis was quantified by macroscopic evaluation of MDA-MB-231 lung nodules in Buijon’s solution on Day 3. Endothelial cells from lungs were isolated using magnetic CD31 beads followed by staining with CD137 or CD276 and sorted using a BD FACS Aria IIu SORP. All studies were conducted as per protocol approved by Harvard IUCAC.
Facs sorp aria iiu
Manufactured by BD
The BD FACS SORP Aria-IIu is a high-performance flow cytometry instrument designed for advanced cell analysis and sorting. It features a simplified user interface, enhanced optical alignment, and a compact footprint. The core function of the BD FACS SORP Aria-IIu is to analyze and sort cells based on their physical and fluorescent characteristics.
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Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Facsaria iiiu, by BD (2 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Axio observer a1 microscope, by Zeiss (1 mentions)
Jetprime transfection reagent, by Polyplus Transfection (1 mentions)
Ghost red 780, by Cytek Biosciences (1 mentions)
Anti cd45 magnetic beads, by Miltenyi Biotec (1 mentions)
Deoxyribonuclease 1, by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Automacs pro separator, by Miltenyi Biotec (1 mentions)
Calcein violet, by Thermo Fisher Scientific (1 mentions)
Ethidium homodimer 1 ethd 1, by Thermo Fisher Scientific (1 mentions)
8 protocols using facs sorp aria iiu
1
Quantifying Lung Metastasis and Endothelial Cell Isolation
2
Quantifying Lung Metastasis and Isolating Endothelial Cells
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MDA-MB-231 or 4T1 cells were injected i.v. into female CD1 nude or balb/c mice (4–6 weeks) respectively. Animals were sacrificed at defined time points and lungs were harvested. Lung metastasis was quantified by macroscopic evaluation of MDA-MB-231 lung nodules in Buijon's solution on Day 3. Endothelial cells from lungs were isolated using magnetic CD31 beads followed by staining with CD137 or CD276 and sorted using a BD FACS Aria IIu SORP. All studies were conducted as per protocol approved by Harvard IUCAC.
3
CellTag Barcoding for Single-Cell RNA-seq
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CellTag barcoding plasmids containing constitutively expressed GFP construct were packaged into lentivirus with a titer of 2.5–3.5 × 108 TU/mL [15 (link),16 (link)]. A 200 μL aliquot of lentiviral Library #1 was added to low-passage naive RB028 cells in a 24-well plate, and the culture was centrifuged for 1 h at 1000× g in 32 °C. Cells were allowed to recover for at least one week. After a GFP signal was observed, cells were manually dissociated over ice with a P1000 pipette (pipetting 30–40 times) and passed through a 30 μm cell strainer. GFP+ RB028 cells were selected using fluorescence-activated cell sorting (FACS) with BD FACS SORP Aria-IIu, reaggregated using anti-adherence-rinsing-solution-treated microwell plates (STEMCELL Technologies, catalogue #34411 and #07010) and recovered overnight with rock inhibitor Y-27632 (10 μM; Selleckchem, catalogue #S1049). Cell clusters were lifted one week later and transferred into a flask for routine cell culturing protocol and then mechanically dissociated for each of the scRNA-seq experiments. In total, 10,000 cells were targeted for capture using 10X Illumina 3v3.1′ chemistry. The remaining cells were reaggregated into microwell plates with Y-27632 overnight. Once the culture recovered, cells were treated with 1 μM carboplatin to generate chemoresistance as described above.
4
Generation and Characterization of FUT8 KO HEK293T Cells
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HEK293T cells (ATCC, Manassas, VA, USA) were transfected with three gRNAs for human FUT8 using JetPrime transfection reagent (Polyplus-Transfection, New York, NY, USA). Cells were examined by GFP fluorescent microscopy at 24 h post-transfection using a Zeiss Axio Observer A1 Microscope to gauge sufficient levels of expression necessary for downstream flow cytometric analysis and cell sorting. Following microscopy, cells were harvested, washed in PBS, and resuspended in DMEM with 1 mM EDTA to prevent the formation of cell aggregates. Cells were sorted on a 5-laser 17-color BD FACS SORP Aria-IIu with an Automatic Cell Deposition Unit (ACDU). The top 20% GFP-expressing cells were individually sorted into a 96-well plate. FSC-W by FSC-A and SSC-W by SSC-A were used to reduce the rate of duplets. 4 h post-sort, cells were inspected to ensure that all wells contained only one cell. Any wells that contained duplets were excluded from further processing. Once clones reached confluence in a 6-well plate, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA) and used for western blot analysis.
Upon acceptance of this manuscript, FUT8 KO cell line clone 4 will be licensed for distribution through the University of Miami Office of Technology Transfer and will be commercially available for purchase.
Upon acceptance of this manuscript, FUT8 KO cell line clone 4 will be licensed for distribution through the University of Miami Office of Technology Transfer and will be commercially available for purchase.
5
Retinal Cell Isolation and Sorting
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Three days after optic nerve crush, retinas were dissociated using papain digestion (17 U/ ml papain, Worthington; 5.5 mM L-cysteine; 0.006% DNase; 1.1 mM EDTA in DMEM/2% B27) during 30 min at 37 °C as previously described at [35 (link)]. After digestion, retinas were washed in DMEM, gently triturated using a Pasteur pipet and centrifuged at 300 g × 5 min at RT. Dissociated cells were resuspended in DMEM/2% B27, passed through a 35 μm cell strainer and placed on ice until use. Dissociated retinal cells were stained with Ghost red 780 (TONBO Biosciences) to exclude non-viable cells and then CFP cells were separated by BD FACS SORP Aria-IIu (BD Biosciences) at Flow Cytometry Shared Resource, University of Miami, Sylvester Comprehensive Cancer Center. C57BL6/J retinal cells were used as unstained control. Sorted cells were collected in PBS and immediately frozen at − 80 °C.
6
Isolation and Characterization of Perivascular Mesenchymal Cells
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For analysis of MSCs, BM cells were isolated by flushing from femora and tibiae and were then enzymatically digested with 7 mg/mL collagenase type I (#17100-017; Invitrogen) and 1 mg/mL deoxyribonuclease I (D5025-750KU; Sigma-Aldrich) in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) at 37°C for 20 min. Cells were collected and washed with PBS +2% FCS and centrifuged at 600 × g for 5 min at 4°C. The cells were resuspended in PBS +2% FCS and filtered through a 70-μm nylon mesh. The cells were then centrifuged again at 600 × g for 5 min and incubated with an anti-CD16/32 antibody (2 μL/mouse) for 5 min at 4°C for Fc receptor blockade. Anti-CD45 magnetic beads (Miltenyi) were then added at a 1/10 vol/vol ratio and incubated for 15 min at 4°C. After unbound antibody was removed by two washes with PBS +2% FCS, CD45+ cells were isolated using an AutoMACS Pro Separator (Miltenyi) with the Depletes-S program. The isolated cells were centrifuged at 340 × g for 5 min and stained with flow cytometry antibodies at 4°C. CD45−Ter119−CD31−LepR+CD140a+ cells were isolated as perivascular mesenchymal cells. Dead cells were eliminated by staining with 1 μg/mL propidium iodide (Invitrogen). Cell sorting was performed using an SORP FACS Aria IIu or FACS Aria IIIu instrument (BD Biosciences). Data were analyzed with FlowJo software (TreeStar).
7
Hematopoietic Stem Cell Subpopulation Analysis
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HSC fractions were analyzed essentially as described previously (37 (link)). HSPCs were phenotypically defined as follows: LT-HSCs, lineage−Sca-1+c-Kit+CD34−Flt3− or lineage−Sca-1+c-Kit+CD48−CD150+; short-term HSCs, lineage−Sca-1+c-Kit+CD34+Flt3− or lineage−Sca-1+c-Kit+CD48−CD150−; MPPs, lineage−Sca-1+c-Kit+CD34+Flt3+; MPP2 cells, lineage−Sca-1+c-Kit+Flt3−CD48+CD150+; MPP3 cells, lineage−Sca-1+c-Kit+Flt3−CD48−CD150−; MPP4 cells, lineage−Sca-1+c-Kit+Flt3+; common lymphoid progenitors, lineage−Sca-1lowc-KitlowIL-7R+Flt3+; megakaryocyte/erythroid progenitors, lineage−Sca-1−c-Kit+CD16/32−CD34−; and granulocyte/macrophage progenitors, lineage−Sca-1−c-Kit+CD16/32+CD34+. Cell sorting was performed using a SORP FACS Aria IIu or FACS Aria IIIu instrument (BD Biosciences). Data were analyzed with FlowJo software (TreeStar).
8
Single-cell isolation and sorting for scRNA-seq
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