Antibodies used for flow-cytometric analysis and fluorescence-activated cell sorting (FACS) of rhesus macaque cells include anti-CD34 (clone 563, BD, Franklin Lakes, NJ), anti-CD45 (clone D058-1283, BD), anti-CD45RA (clone 5H9, BD), and anti-CD90 (clone 5E10, BD). Antibodies were used according to the manufacturer recommendation. Dead cells and debris were excluded via forward scatter/side scatter gating. Flow-cytometric analyses were performed on an Symphony I and FACSAria IIu (BD). Cells for in vitro assays, as well as NHP stem cell transplants, were sorted using a FACSAria IIu cell sorter (BD), and purity was assessed by recovery of sorted cells. CD34+CD90+CD45RA− sorting for transplantation was performed in yield mode to increase cell recovery, whereas cells for colony-forming cell (CFC) assays were sorted in purity mode to prevent crosscontamination by different subsets.
Facsaria iiu
Manufactured by BD
Sourced in United States, United Kingdom, Germany
The FACSAria IIu is a high-performance cell sorter designed for advanced flow cytometry applications. It features multiple lasers and detectors for comprehensive cell analysis and sorting. The core function of the FACSAria IIu is to enable efficient and accurate cell sorting based on user-defined parameters.
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Lab products found in correlation
Lsrfortessa, by BD (9 mentions)
Sytox blue, by Thermo Fisher Scientific (5 mentions)
Facsaria fusion, by BD (5 mentions)
Facscalibur, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (4 mentions)
Propidium iodide, by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Facsaria iiu cell sorter, by BD (4 mentions)
Trustain fcx, by BioLegend (3 mentions)
Facsdiva, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Fortessa x20, by BD (3 mentions)
Fortessa, by BD (3 mentions)
Ficoll paque plus, by GE Healthcare (3 mentions)
Lsr iiu, by BD (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Facsaria 3, by BD (2 mentions)
239 protocols using facsaria iiu
1
Flow Cytometry and Cell Sorting of Rhesus Macaque Cells
2
Multiparametric Flow Cytometry Analysis
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Cells were isolated as described above and suspended in HBSS containing 2% FBS and 0.2% sodium azide. Cell suspensions were incubated with 50 µl of purified mAbs against mouse CD16/CD32 (2 µg/ml) and fluorochrome-conjugated antibodies for 20 min on ice. Before flow-cytometric analysis, cells were suspended in 0.5 µg/ml propidium iodide solution to distinguish between live and dead cells. For intracellular staining, cells were fixed and permeabilized with a Foxp3 Staining Set (eBioscience) or IntraPrep Permeabilization Reagent (Beckman Coulter) according to the manufacturer’s protocols. Cells were analyzed using a FACSCalibur, FACSAria IIu, or FACSAria III (BD) and sorted using a FACSAria IIu or FACSAria III. All data were analyzed using FlowJo software (TreeStar). Within each experiment, we used several types of lineage markers. To clearly present the gating strategies, the lineage markers and pregating lists are provided in Table S1.
3
Circulating Tumor Cell Enrichment Protocol
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Initially, CTCs were enriched through density gradient centrifugation coupled with flow cytometry. Briefly, 4 mL of whole blood was diluted with 4 mL of phosphate-buffered saline (PBS) and gently layered into a centrifuge tube (Corning Inc, Corning, USA) containing 4 mL Ficoll-Paque Premium sterile solution (GE-Healthcare, Uppsala, Sweden). After density gradient centrifugation (1,040 × g, 20 °C, 15 min) without breaking, the interphase consisting of peripheral blood mononuclear cells and CTCs was transferred to a clean tube and washed using PBS. Red blood cell lysis was performed using ammonium-chloride-potassium (ACK) lysing buffer (Boster Biological Technology, Ltd, Wuhan, China). Subsequently, the enrichment was performed by incubating with CD45-PE-Cy5 (#555484; Becton Dickinson Pharmingen, San Diego, CA, USA) and isotype control (#555750; Becton Dickinson Pharmingen) antibody, and CTCs were negatively enriched via hematopoietic cell (CD45+) depletion through flow cytometry (BD FACSAria™ IIu; Becton Dickinson). During flow cytometry, the main cell population was gated based on their forward and side scatter properties to avoid the interference of the cell debris. In the gating strategy, isotype control for each blood sample was used to gate the PE-Cy5 negative area.
4
Isolation of Colonic Macrophages in Rats
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Cell sorting was performed using a BD FACSAria IIU instrument (Becton Dickinson, Franklin Lakes, NJ). Mucosa/submucosa tissue from the entire colon of each rat was dissociated in 10 mL Hanks’ balanced salt solution containing 0.1 mg/mL Liberase (Sigma) and 0.1 mg/mL DNase I, and passed through 70-micron filters. The associated macrophages were isolated by sorting CD163+ and propidium iodide–negative cells (viable cells) from a 40% Percoll fractionation of dissociated mucosal cells obtained from 4 groups of rats, euthanized 7 days after AI. This time point provided the optimal isolation of mature tissue macrophages, based on pilot studies (not shown). Expression of CD163, a member of the scavenger receptor cysteine-rich family class B, was restricted to cells of the monocyte/macrophage lineage, and it is expressed in most subpopulations of mature tissue macrophages.
5
Flow Cytometry Analysis of CXCR4 and EGFP
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Cells were prepared for FACS by removal from plates/wells with Accumax (Innovative Cell Technologies Inc.) according to the manufacturer's instructions. Staining for CXCR4 surface marker was performed using APC anti-human CD184 (CXCR4; BioLegend) or with APC Mouse IgG2a, k isotype control (BioLegend) following the instructions from the manufacturer. The cells were finally passed through a 40 μm strainer then kept on ice before sorting with BD FACS Aria IIU (Beckton Dickinson) with assistance from core facility operator Mark Griffin. For analysis of EGFP levels in NKX2.5→EGFP cells, cells were detached with 1x Trypsin/EDTA (0.25%; Thermo Fisher) according to the manufacturer's instructions and either measured immediately or fixed with 2% paraformaldehyde dissolved in PBS if measured later on the Guava EasyCyte HT (Luminex) flow cytometer. For all flow/FACS applications, gating on relevant forward and side scatter parameters was performed to restrict any events not identified as single cells. FlowJo software was used for data analysis.
6
Isolation and Sorting of PBMCs
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PBMCs were isolated by gradient centrifugation using Gradisol L (Aqua Medica, Lodz). Isolated PBMCs were stained with monoclonal antibodies anti-CD3 PE-Cy7 and anti-TCRγδ FITC (Supplementary Table S2 ). After 20 min of incubation in the darkness at room temperature, the excess of antibodies was washed out with PBS. Samples were directly sorted with BD FACS Aria IIu (Becton Dickinson, Franklin Lakes, NJ, USA). The full specification is presented in Supplementary Table S1 . Each time after sorting, the purity of cells was assessed. Samples were processed further only if the purity was >95%. Sorted cells immediately after purity assessment were suspended in the RLT buffer (Qiagen, Inc., Valencia, CA, USA) with β-mercaptoethanol and frozen at −80 °C. The gating strategy is presented in Supplementary Figure S2 .
7
High-Throughput Flow Cytometry Screening
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Cells
were analyzed and
sorted with a BD FACSAria IIu flow cytometer (Becton Dickinson (BD),
Franklin Lakes, NJ). A blue solid-state laser (488 nm excitation)
and a 530/30 nm filter was used to measure eGFP. FCS files were analyzed
using Flowing Software v2.5.1 (Cell Imaging Core, Turku Centre for
Biotechnology, Turku, Finland). For flow cytometry sampling, the geometric
mean of the FITC-A fluorescence for 10 000 events was taken
as the “promoter activity”. Prior to sorting, the cytometer
was calibrated using Accudrop beads (BD) and SPHERO Rainbow calibration
particles (Spherotech, Lake Forest, IL).
On the day of sorting,
2–4 mL of library frozen stocks were thawed, centrifuged to
remove excess glycerol, and used to inoculate 25 mL of MOPS minimal
medium supplemented with 0.4% (w/v) xylose, 100 μg/mL ampicillin,
and 0.1 mM IPTG. Cells were monitored hourly until they reached a
postrecovery state (∼5 h), as indicated by 85–90% of
cells expressing GFP. Cells were then dosed with 0 or 100 μM
formaldehyde and sorted 2 h later (Figure S8 ). The final promoter library in the NEB5α and ΔfrmR strains was sorted into eight gates with approximately
equivalent populations, and 1 000 000 events were collected
from each gate directly into LB medium. Populations were recovered
at 37 °C overnight and miniprepped for sequencing. Plating indicated
that approximately 70% of the sorted cells survived.
were analyzed and
sorted with a BD FACSAria IIu flow cytometer (Becton Dickinson (BD),
Franklin Lakes, NJ). A blue solid-state laser (488 nm excitation)
and a 530/30 nm filter was used to measure eGFP. FCS files were analyzed
using Flowing Software v2.5.1 (Cell Imaging Core, Turku Centre for
Biotechnology, Turku, Finland). For flow cytometry sampling, the geometric
mean of the FITC-A fluorescence for 10 000 events was taken
as the “promoter activity”. Prior to sorting, the cytometer
was calibrated using Accudrop beads (BD) and SPHERO Rainbow calibration
particles (Spherotech, Lake Forest, IL).
On the day of sorting,
2–4 mL of library frozen stocks were thawed, centrifuged to
remove excess glycerol, and used to inoculate 25 mL of MOPS minimal
medium supplemented with 0.4% (w/v) xylose, 100 μg/mL ampicillin,
and 0.1 mM IPTG. Cells were monitored hourly until they reached a
postrecovery state (∼5 h), as indicated by 85–90% of
cells expressing GFP. Cells were then dosed with 0 or 100 μM
formaldehyde and sorted 2 h later (
equivalent populations, and 1 000 000 events were collected
from each gate directly into LB medium. Populations were recovered
at 37 °C overnight and miniprepped for sequencing. Plating indicated
that approximately 70% of the sorted cells survived.
8
Immunophenotyping of Stem Cells
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Immunophenotypic analysis was performed using flow cytometry. For this, 1 × 105 cells/ml were trypsinized, centrifuged and fixed in 10% paraformaldehyde. The cells were then incubated with primary antibodies (dilution 1:100) for 30 minutes at 4 °C. After this period, the cells were incubated with the secondary antibody (dilution 1:500) for 30 minutes at 4 °C. Finally, the cells were washed and analysed by a BD FACSariaIIu flow cytometer (Becton Dickinson, San Jose, CA, USA). Controls were performed using unmarked cells exposed only to the non-specific secondary antibody. For each sample, 10,000 events were counted. The primary antibodies used were as follows: Nanog (n-17, sc30331), Sox-2 (sc-17320), vimentin (sc-73259), cytokeratin 18 (RGE53, sc-32329), Stro-1 (sc-47733), PCNA-3 (sc-46), β-tubulin (sc-47751), SSEA-4 (sc-59368), TRA-1-60 (sc-21705) and CD73 (sc-14684) (all purchased from Santa Cruz Biotechnology, Inc., Europe); in addition to CD105 (ab53321; Abcam, Cambridge, UK), CD117 (A4502; Dako Cytomation, Carpinteria, CA, USA) and CD45 (MHCD4501; Invitrogen, Carlsbad, CA, USA). For nuclear antibodies such as PCNA-3, Nanog and Sox-2 we added 0.1% of triton (catalogue number 13-1315-05; LGC Biotecnologia) in order to permeabilize the cell membranes.
9
Identification of CXCR4 and CD99 Positive Cells
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Cells were removed from plates/dishes by scraping into phosphate buffered saline (PBS), then an equal volume of Accumax (Innovative Cell Technologies Inc., San Diego, CA, USA) was added. This was incubated 5 to 10 min at room temperature, at which time dissociation to a single cell suspension was verified by microscopy. Surface marker staining was performed to identify CXCR4 and CD99 positive cells following the manufacturer’s instructions. The antibodies used were APC mouse anti-human CD184 (CXCR4; clone 12G5, BioLegend, San Diego, CA, USA), APC mouse anti-human CD99 (clone HCD99, BioLegend), or APC Mouse IgG2a, k isotype control (catalog # 400222, BioLegend). After staining and washing, the cells were passed through a 40 µm strainer and kept on ice in the dark before analysis with BD FACS Aria IIU (Beckton Dickinson, Franklin Lakes, NJ, USA) with assistance from the core facility operator. Data were analyzed using FlowJo software.
10
Lentiviral Transduction of Endothelial Cells
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To monitor angiogenesis, EC were transduced to express GFP or dTomato. The GFP- or dTomato-expressing plasmids pHR’SIN-cPPT-SEW, together with pCMV-DR8.91 and pMD.G (all kindly provided by Prof. Patrick Maier, Department of Radiation Oncology, University Medical Centre Mannheim, Germany), were used to produce lentiviral vectors through the transient transfection of 293FT cells. The HUVEC and HRMVEC were transduced once in the presence of polybrene (8 μg/ml; Sigma-Aldrich). The transduced GFP- or dTomato-positive EC were sorted using a BD FACSAria IIu (Becton Dickinson, Heidelberg, Germany), collected, and cultured as reported previously. The transduced EC were used from passages 5 to 11.
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