For cell surface marker staining, cells were collected, washed once with PBS supplemented with 2% FCS (FACS buffer). Cells were incubated with anti CD34 (#343516581), CD90 (#3281145E10) and EPCR (#351906) (BioLegend) antibodies for 30 minutes (min) at 4°C, and washed once with cold FACS buffer. For cell sorting, CD34+ cells were quickly thawed and stained for CD34, CD38 (#345806), CD45RA (#560362) (BD Bioscience) and CD90 following the same procedure as above. When specified, cells were stained with the Annexin V Apoptosis Detection Kit, according to the manufacturer’s protocol (BD Bioscience). All data were collected on FACS Canto II or LSRII analyzer (Becton Dickinson), and analyzed with FlowJo software. Cells were sorted on a FACS Aria II or III (Becton Dickinson).
Facs aria 2 or 3
Manufactured by BD
The BD FACS Aria II or III is a high-performance cell sorter designed for precise cell separation and analysis. It features advanced optics and fluidics systems to enable accurate sorting of complex samples. The core function of this instrument is to provide efficient and reliable cell sorting capabilities for research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Annexin 5 apoptosis detection kit, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Automacs, by Miltenyi Biotec (2 mentions)
Macs system, by Miltenyi Biotec (2 mentions)
Facscalibur, by BD (2 mentions)
Efluor 780, by Thermo Fisher Scientific (2 mentions)
Foxp3 fixation permeabilization kit, by Thermo Fisher Scientific (2 mentions)
Symphony s6, by BD (2 mentions)
Facsymphony, by BD (2 mentions)
Efluor 506, by Thermo Fisher Scientific (2 mentions)
Anti cd34, by BioLegend (1 mentions)
Lsrii analyzer, by BD (1 mentions)
7 aminoactinomycin d 7 aad, by Merck Group (1 mentions)
Ly6g 1a8, by BioLegend (1 mentions)
Cd115 clone afs98, by Thermo Fisher Scientific (1 mentions)
Cd19 clone 1d3, by BD (1 mentions)
Cd11b clone m1 70, by BD (1 mentions)
Cd11c clone n418, by BioLegend (1 mentions)
7 aad, by BD (1 mentions)
22 protocols using facs aria 2 or 3
1
Immunophenotypic Characterization of Cells
2
Multiparametric Flow Cytometry Analysis
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Before analysis or sorting, cells were labeled with different combinations of the following fluorochrome–conjugated antihuman antibodies: CD14 (HCD14), CD19 (HIB19), CD3 (UCHT1), CD33 (P67.6), CD34 (8G12), CD38 (HIT2), CD45 (HI30), CD45RA (HI100), CD49f (GoH3), CD90 (5E10), CD133 (13A4), and CD71 (L01.1). Dead cells were excluded with 7-aminoactinomycin D (7AAD, Sigma Aldrich). Apoptotic cells were stained with the Annexin V apoptosis detection kit, according to manufacturer’s protocol (BD Bioscience). All data were collected on fluorescence-activated cell sorter (FACS) Canto II or LSRII analyzer (Becton Dickinson) and analyzed with FlowJo software. Cells were sorted on a FACS Aria II or III (Becton Dickinson).
3
Multicolor Flow Cytometry Immunophenotyping
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For flow cytometry or cell sorting, cells were transferred to 15-mL tubes and washed in cold buffer (2% FCS, 2 mM EDTA in PBS). Before antibody labeling, cells were incubated with Fc-Block (1:200, anti-CD16/32 antibody; 2.4G2, BD Pharmingen) for 10 min at 4°C and washed. Cells were stained in buffer with (1:100) fluorescently labeled antibodies against CD11b (clone M1/70, BD Pharmingen), CD11c (clone N418, Biolegend), CD115 (clone AFS98, eBioscience), CD19 (clone 1D3, BD Pharmingen), and Ly-6G (1A8, Biolegend) for 30 min at 4°C in the dark. 7-AAD (BD Pharmingen) was added before measurement to exclude dead cells. Analysis was performed on LSRFortessa and sorting on FACSAria II or III (BD Pharmingen). Unstained, empty vector-transduced cells or fluorescence-minus-one staining setups served as controls.
4
Isolation and Analysis of Lymphocyte Subsets
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Single cell suspensions of spleen, mesenteric lymph nodes (mLNs), or various subcutaneous lymph nodes (scLN) (Schallenberg et al., 2010 (link)) were prepared using 70 μm cell strainers (BD Biosciences). mAbs to CD4 (RM4–5, GK1.5), CD25 (PC61, 7D4), CD62L (MEL-14) and Thy1.1 (OX-7), as well as Fc receptor–blocking mAbs to CD16/32 (93) and Pacific Blue- and APC-conjugated streptavidin were obtained from eBioscience or BD Biosciences. The mAb to TCR-HA107–119 (6.5) was purified and conjugated to Alexa Fluor 647 (Invitrogen) in our laboratory according to standard protocols. Before FACS, for some experiments, CD4+ CD25+ cells were enriched using biotinylated mAbs against CD4 or CD25, respectively, streptavidin-conjugated microbeads and the AutoMACS (Miltenyi Biotec). Samples were analyzed on a LSR II or sorted using a FACSAria II or III (BD). Data were analyzed using FlowJo (Tree Star, Inc.).
5
NP-Ficoll Immunization of Transferred B Cells
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1 × 106 CD45.1 B1-8hi naive B cells were transferred into B6 mice, which were then immunized i.v. with NP-Ficoll on the next day. Donor B cells were magnetically enriched from pooled spleens of recipient mice using APC-conjugated anti-CD45.1 and anti-APC MicroBeads (130-090-855; Miltenyi Biotec), with the MACS system (Miltenyi Biotec). After enrichment, cells were further stained with respective Abs and sorted using FACSAria II or III (BD Biosciences) as shown in Figure 4—figure supplement 1A . Rv-transduced donor B cells were enriched as described above and sorted as shown in Figure 4—figure supplement 1C .
6
Purification of 2C-like and ESCs by FACS
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Cells were washed with room temperature sterile PBS, trypsinized and re-suspended in ice-cold sterile 0.5% BSA PBS solution. Sorting was performed using a BD BioSciences FACS Aria II or III. During sorting, cells were collected in culture medium and kept at 4 °C during the sort. Analysis of FACS data was performed using the FlowJo software and the same gatings were used for all replicates of a same experiment. Cells were not index sorted and the purity of the sort was estimated at 96% for 2C-like and 97% for ESCs. A control flow profile for wt ESCs is shown in Supplementary Figure 1c . For the Rex1 experiments, the Rex1- gate was defined based on the fluorescence of WT ESCs and the Rex1high gates were defined based on the fluorescence of Rex1-GFP cells cultured in 2i. FACS Calibur (BD Biosciences) was used to quantify the population of eGFP-positive cells. Cells that had been frozen in 2i were thawed and the Rex1 sorting was performed 4 days after 2i withdrawal. For data presented in Figure 7a , the 2C::turboGFP and Zscan4c::mCherry cell line was FACS sorted just before transfection and the 2-cell-like cells or Zscan4::mCherry positive cells were removed respectively from the population.
7
Purification of 2C-like and ESCs by FACS
8
Sorting and Isolation of Colon Immune Cells
9
Intestinal Lymphocyte Isolation and Analysis
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Lymphocytes from lamina propria (LP) and intraepithelium were isolated as described above. Peyer’s patches (PP), inguinal lymph nodes (iLN), mesenteric lymph nodes (mLN), and spleen (Spl) were isolated, and erythrocytes were lysed with ACK lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA). Single cell suspensions were stained with primary antibodies for CD3, CD8α, CD8β, CD62L, CD103, TCRγδ, TCRβ, B220, CD11b, and F4/80. To evaluate IgA-producing cells, cells were stained with biotin-conjugated anti IgA antibody, followed by Streptavidin-PE. In some experiments, IELs (CD3+CD8α+TCRβ+ or TCRγδ+ cells) and intestinal epithelial cells (CD103-EpCAM+ cells) were sorted by a FACS Aria II or III (BD Biosciences) from isolated intestinal epithelium. All analyses were performed on a FACS Canto II (BD Biosciences) with FlowJo Software (Tree Star).
10
Immunophenotyping of Hematopoietic Stem Cells
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HSC (CD150+ CD48- Lin- Sca-1+ c-Kit+) or HSPCs (Lin- Sca-1+ c-Kit+) were sorted by FACS AriaII or III (BD) and fixed with 4%PFA. Cells were permeabilized with 0.5% Triton for 15min and stained with anti-Tomm20 (ab78547 or ab289670) 1:200 or anti-ssDNA (18731, IBL) 1:50. Secondary stains were performed with anti-rabbit-AF488 1:1000 or anti-rat-AF488 1:1000. DAPI 1:1000 or 7-AAD 1:200 were used for nuclear staining. Data correction was performed with Mark-II (Amnis) and analysis was performed with IDEAs software (Amnis).
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