2.1. Set up a flow cytometry staining panel for your populations of interest. Protocols - from cell preparation to analysis OVERVIEW The following protocols are included in this document: 1. On day 5, cells were harvested and the CFSE fluorescent staining was analyzed by flow cytometry. Antibodies to (v) (CD51; BD Biosciences and BioLegend) and (3) (CD61; BD Biosciences and BioLegend) were used to both detect or block the surface-bound molecules on responder T cells.
All protocols described in this article have been tested using at least three hiPSC lines and demonstrated as highly reproducible (Cao et al., 2019). but it is applicable as a consistent parameter for the CFSE-labeling and -detection protocol optimization procedure. CFSE Staining. 2.
CFSE is a non-fluorescent molecule that easily diffuses across cell membranes. Expansion and treatment of . Intracellular and Foxp3 staining were performed according to the manufacturer's protocols and cell samples were then used for flow cytometry. In the second edition of this series, we described the use of cell tracking dyes in combination with tetramer reagents and traditional phenotyping protocols to monitor levels of proliferation and cytokine production in antigen-specific CD8 + T cells. MojoSort Isolation Kits typically contain a biotin-antibody cocktail and Streptavidin Nanobeads, intended to isolate an untouched cell population. 4.7/5.0 (3 reviews) BioLegend develops world-class, cutting-edge antibodies and reagents for biomedical research, manufactured at its state-of-the-art facility in San Diego, CA. CFSE is widely used for cell tracking and proliferation studies. 3. MATERIALS: A. CFSE is toxic to cells. (B) Chemical structure of CFDA-SE. BioLegend. used in our staining protocols. Prepare stock solution by reconstituting 1 vial of lyophilized CFSE dye in 36 L of DMSO to make a 5 mM solution. During proliferation, CFSE is equally distributed among the "daughter cells", whereas the overall fluorescence in each proliferating cell decreases. Stem Cells Immunology BioLegend is the leader in creating complete research solutions for immunologists. To isolate immune cells from a mouse spleen before sorting Before starting Dissect spleen from mouse 1 Prepare 50mL of 10 FBS RPMI media 5 mL FBS. Techniques: In Vitro, In Vivo, Incubation, FACS, Lysis, Staining, Inhibition, Mouse Assay, Injection Briefly, PBMCs were resuspended with carboxyfluorescein succinimidyl ester (CFSE) in PBS (final concentration, 1M) at 1 10 7 cell/mL and incubated for 5 minutes at 37C in the dark. Tissue will be sectioned and preserved in -20 C before use. CFSE is a versatile tool for the fluorescent intracellular labeling of live cells. Cells were gated on a . [Abstract] Proliferative capacity and degranulation are important features of antigen-specific CD8+ T cells. Therefore, CFSE staining can be used as a quick screening tool for cellular proliferation of virtually . Inside the cell, acetate groups are cleaved by intracellular esterases yielding a fluorescent molecule whose succinimidyl ester group covalently interacts with primary amines of intracellular proteins. BioLegend's mission is to accelerate research and discovery by providing the highest quality products at an outstanding value, along with superior customer . ZERO BIAS - scores, article reviews, protocol conditions and more . 2.4. 7-AAD is a membrane impermeant dye that is generally excluded from viable cells. The results were expressed as the percentage of cells above the negative region for each . Antibody staining . Optimal CFSE labelling reaction setup was sought by testing different conditions (0.625, 2, 8 and 30 M CFSE) using 2.5 5 10 6 cells/mL PBMC, and reaction time set at 5-10 min at RT and quenched by adding 5-10 volumes of either "staining" or "expansion" medium before spinning the cells down and resuspension in pre-warmed . CFSE is compatible with standard intracellular staining protocols . Keir ME, Butte MJ, Freeman GJ, Sharpe AH. Western blot analysis of inflammationassociated protein expression. For Research Use Only. Universit de Sherbrooke Hello Shuoyang, As you can see, the protocol is relatively simple. Protocol. Prepare single cell suspension of peripheral blood mononuclear cells (PBMC) or purified T/NK cells. Follow the steps from 1 to 6 in Subheading 3.1. n = 19 donors.
Isolated cells were stained with CFSE staining reagent (BioLegend, San Diego, CA, USA) according to manufacturer's protocol. (C) . If a final concentration of 5 uM is desired, add .5 uL of the 10 mM reconstituted solution . BrdU labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells within a living animal. I. CFSE staining time of 5 min, which resulted in the highest CDI, was selected to provide the best balance between cell death, background proliferation, and antigen-specific proliferation. Zombie Aqua Fixable Viability kit mouse IL-17A ELISA kit, and carboxyfluorescein succinimidyl ester (CFSE) were also from Biolegend . washed with PBS, re-suspended in RPMI with 1% FBS and labeled with 15 M CFSE (Life Technologies) for 10 min at 37C. . the cells were stained by FITC anti-mouse CD4 (Catalog #100509) according to the Cell Surface Immunofluorescence Staining Protocol (BioLegend), followed by staining with Alexa .
(BioLegend, BD Biosciences, eBioscience) for 20 min at 4C. Usually, CFSE dye may be traced through 6-8 generations by flow cytometry. BioLegend: Cat#301049: Purified anti-human CD28: BioLegend: Cat#302934: HRP anti-human 2-microglobulin antibody: 480019). FITC / AlexaFluor 488 / GFP / CFSE / YFP: A: 655 LP: 695/40 BP: PerCP-Cy5-5 / PerCP--eFluor 710 / PI: Yellow-Green . . Tissue sections will be air dried 15 minutes before 15 minutes cold acetone fixation. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. 6. Spin down and resuspend cells at 10-100 x 10 6 cells/mL in the CFSE working solution. Here, we provide a general protocol for the production of MHC tetramer. Bioprocess forces and protocols will expire shortly. PBMCs were labeled with a cell tracker (CFSE Cell Division Tracker Kit, BioLegend) in accordance with the manufacturers protocol. Superior performance bright, single-peak staining enables visualization of multiple generations The provided CFSE is sufficient for ~1000 assays. No. Further those monomers compose tetramer to stain antigen-specific CD8 + T cells. Article Snippet: In vitro ADCC assay on CT26A33.C3 and C51A33.A5 cells CT26A33.C3 and C51A33.A5 were stained with CFSE dye (BioLegend) as described by the manufacturer's protocol and seeded in a 48-well plate (2104 /well). 7-AMINO-ACTINOMYCIN D STAINING OF DEAD CELLS FOR FLOW CYTOMETRY 7-Amino-actinomycin D (7-AAD) intercalates into double-stranded nucleic acids. Preparation of splenocytes 2. (A) Representative gating strategy. 2. CFSE- 2 D. QIN ET AL. Cell activation analysis 6. We recommend our MojoSort Buffer (Cat. The levels of the inflammationassociated proteins Using our protocol, functional monocytes and polarized macrophage subtypes can be efficiently derived from hiPSCs within 2 and 3 weeks, respectively. Cell staining and flow cytometry For surface staining, single-cell suspensions of lung cells, BM cells, and splenocytes were washed with cell-staining buffer (BioLegend) and stained with anti-Gr1, anti-CD11b, anti-CD11c, anti-F4/80, anti-CD4, anti-CD90.2, and anti-lineage cocktail antibodies for 30min at 4C. as detected by intracellular staining (Figure 2A) or ELISA . 3. If cells are obtained from a buffy coat or whole blood, proceed as . I would use couple of concentration, incubate at 37 for about 5 min, and. (A) Flow cytometry analysis of Sp2/0 mouse myeloma cells labeled with CFDA-SE and analyzed by flow cytometry after being cultured for 0, 24, and 48 hours (filled histograms). CFSE staining and cell culture. CFSE Labeling Procedure: 1. panel; BioLegend, Inc.) and the levels of Th1 and Th2type cytokines (cat. Cell count analysis 7. To use these products, you also need a separation buffer and a magnetic separation system. The protocol was performed as previously described (24,25). The reaction was then quenched with 10 volumes of complete media (RPMI . 740405; BioLegend, Inc.) were measured by flow cytometry. Figure 2: Overview of the uEVs staining and capture in the cytometer. During the BrdU assay, BrdU is incorporated into replicating . BioLegend: 127407: Chemicals, peptides, and recombinant proteins: Collagenase I: Thermo Fisher Scientific: . Apoptosis is a form of programed cell death that is vital for all multicellular organisms to ensure proper development. We use eBioscience Fixable Viability Dye eFluor 780 which has an excitation of 633 nm and emission of 780 nm.
Yanyu Wang from the Clinical . Apoptosis Assay from BioLegend. To analyze the roles of Bach2 on alternative cell-fate choices at the pre-GC stage, we analyzed the differentiation of transferred B1-8 hi wild-type and Bach2-deficient cells under competitive conditions (Figure 1A).Although donor non-NP-binding Bach2-deficient B cells showed a slight reduction in homing to the spleens, they had no competitive disadvantage when compared with their wild-type . 1.5 Pellet the cells by centrifugation and resuspend them in fresh pre-warmed complete culture medium. and analyzed by standard flow cytometry staining protocols (see section "Flow cytometry"). Anti-human and anti-mouse concentrations obtained after staining the 100% human and mouse samples with their corresponding isotype controls were used to establish the background concentrations of . Aliquots of 50 l should be stored at 20 C and are stable for several months. Protocols for Publication: Flow Cytometry -Based Immune Assessment .
Antigen retrieval helps both the accessibility of . A brief wash with complete medium will then quench any dye remaining in solution. Wash cells twice in sterile 1 X PBS solution to remove serum and re-suspend the cells in room temperature PBS at 1-10 x 10 6 cells/mL. H&E staining will be performed according to general protocol from formaldehyde fixed and paraffin wrapped samples.
BSA solution To make 10% BSA solution, dissolve 2 mg BSA powder in 20 mL ddH 2 O, Sterilize the solution with 0.22 m filter and store at 4C for up to 6 months. Once inside the cells, intracellular esterases cleave the acetate groups to yield the fluorescent carboxyfluorescein molecule. Company: BioLegend. Data shown are representative of three individual . The cells were washed with cell staining media (PBS without Ca2+/Mg2+, 2% bovine . 7-Amino-actinomycin D (e.g., Cat #129935, Calbiochem, San Diego, CA) B. Prior to the initiation of the differentiation . Cells were washed with FBS and twice with TCM. Surface staining of mouse splenocytes and peripheral blood.
. This protocol can also be performed using alternative mouse models (e.g., CD45.1 hosts and CD45.2 recipients) or biochemical tools (CFSE or CTV labeling) to identify transferred monocytes and their progeny in recipient tumors. Measure T cell cytotoxicity after co-culture for 6 h using the 7-AAD/CFSE cell-mediated cytotoxicity assay kit. CFSE Cell Division Tracker Kit by BioLegend. Cells were seeded at a concentration of 1x106cells/ml in 96 well plates in growth medium supplemented with 1g/ml anti-CD3 and . CFSE dilution was used as an indicator of cell proliferation.
Antibody: anti-CD25-PE and anti-CD25-PerCP/Cy5.5 from BioLegend - no . Cells were then surface stained, fixed, and permeabilized using Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer's protocol. CFSE staining (optional) 4. no. PBMCs were labeled with fresh 1 M carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes) at 37C for 2 minutes and then at room temperature for 7 minutes in the dark. Multicolor flow cytometry panels A. FOXP3 FMO FULL STAIN FMO . Immunofluorescent staining will be performed on cryopreserved samples. For cell viability analysis, cells were stained with 7AAD (BioLegend) for 5 min before running on a flow cytometer. ** Re . 2.6 | In vitro proliferation assay Expanded T cells from albino B6 mice were adjusted to 107/mL and labeled with 10 M CFSE (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 10 minutes at room temperature. The panel used in this protocol is shown . CFSE dilution by T cells was analyzed by flow cytometry at . For Research Use Only. For IL-10 intracellular staining, T cells were stimulated with cell stimulation cocktail (eBioscience, UK) for 6 hours and Golgi plug (eBioscience, UK) for 5 hours, prior to intracellular staining using Permeabilizing Solution 2 (BD Not for use in diagnostic or therapeutic procedures. Anti-CD3 staining was used to differentiate T cells from tumor cells. . In total, 500,000 CD4 + cells/well were then plated into 48-well tissue culture plates precoated with 10 g/mL anti-mouse CD3E (BioLegend, catalog 100302) and 2 g/mL anti-mouse CD28 (BioLegend, catalog 102102) in PBS. We also tested manufacturer's labeling protocol for CFSE concentrations of 0.2 M to 20 M, as well as another CFSE supplier (Biolegend #422701, San Diego, CA, USA). However, perhaps there is an excessive or an insufficient amount of apoptotic activity. the manufacturer's protocol. Zombie Aqua Cell Viability Stain, supplied by BioLegend, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. Figure 2 shows a representative protocol to uEVs stain. Subsequently, the cells were washed and resuspended in 1640 Medium for 10 min to stabilize the CFSE staining. Arrange the slides in a staining container. Incubate cells for 20 minutes at room temperature or 37C and keep protected from light. CFSE Cell Division Tracker Kit is composed of 5 vials, 100 g per vial of CFSE (formally known as 5- (and 6)-Carboxyfluorescein diacetate succinimidyl ester of CFDA SE), and 500 l of anhydrous DMSO. Critical: CFSE has an excitation of 492 nm and emission of 517 nm, stain the cytotoxicity assay using a fixable viability dye which does not overlap. So far we have been unable to get any specific CD25 stain. Guidelines for the Use of Flow Cytometry and Cell Sorting in Immunological Studies - Valuable resource for all things flow cytometry - protocols, guidelines, sample preparation etc. Inside the cell, its acetate groups are cleaved by . Thus, CFSE labeling can be applied to estimate the number of the generation after rapid cell proliferations. Migration to media only is shown as negative control. 2. After removal of DCA, CFSE-labeled T cells were co-cultured with DCA treated-Raji cell at a 1:1 ratio for 72 h in the presence of anti-CD3/CD28 antibody. For ICS with tetramer staining, follow step 17 in Subheading 3.1 after surface staining. By combining tetramer staining with a CFSE staining, we were able to enumerate the total number of antigen-specific T cells, as well as their number of divisions upon antigen-specific stimulation during a week. CFSE readily crosses intact cell membranes. CFSE Staining Canada). BioLegend: CAT#100236: CD8 (63-6.7) BioLegend: CAT#100732: CD4 (GK1.5) BioLegend: CAT#100408: APC IFN- (XMG1.2) . 3.3 CFSE Dilution. Improved CFSE Alternatives for Cell Proliferation Permanently label cells with Invitrogen CellTrace fluorescent stains without affecting morphology or physiology to trace generations or divisions in vivo or in vitro.
1 X PBS with Ca2+ and Mg2+ The isolated cells were stained with 5 M CFSE (BioLegend, catalog423801) in PBS following the manufacturer's protocol. 7-AAD can be excited at 488 nm with an argon laser. Protocols; Advanced Concepts. The aim is to stain human gingival epithelial cells with this occludin antibody using immunofluorescence. For those interested in running the protocol in real time, we provide a timer that is pre-set with the appropriate count down time for that step. Not for use in diagnostic procedures. Home > Search Results . Pre-enrichment of T cells (optional) 3. Here, we describe a simple protocol to CFSE-label human T and/or NK cells and analyze their proliferation in vitro by flow cytometry. Finally, the cells . For Ki67 assay, cells (1 10 5 /well) were seeded in 6-well plate, and 2 days later the cells were harvested and did according to the protocol of Ki67 Cell Proliferation Kit (Miltenyi Biotec Inc., Auburn, CA, USA). Prepare a 5 mM stock solution of CFDA-SE (MW 557.47 g/mol) by dissolving 100 g of CFDA-SE in 36 l of anhydrous DMSO. . BD Phosflow Protocols for Mouse Splenocytes or Thymocytes.
CD3, CD4, and (after permeabilization) FOXP3 antibodies work fine. 1.6 Incubate the cells for at least 10 minutes before analysis to allow the CellTrace Violet to T-cell proliferation was assessed by labeling of CD8 + T cells with CFSE (CellTrace); equal volumes of CFSE (2 mol/L in PBS) and 1 10 7 CD8 + T cells/mL in PBS were mixed, the reaction was incubated for 1 minute in a 37C water bath, and cells were then washed twice with TCM.
pdp. The CFSE labeled and unstimulated cells were used as control.
(H5C6), and anti-human CD81 Antibody (5A6), all labeled with phycoerythrin (PE) and purchased from BioLegend (San Diego, CA, USA). Human mesenchymal stromal cells (MSCs) can potently suppress immune responses in vitro and in animal models of human disease (1, 2), but to date MSC-based therapies have produced mixed results in clinical trials for treatment of inflammatory and autoimmune diseases (3, 4).A major challenge in the development of consistently effective MSC-based immunosuppressive therapies is that MSC lines . FOXP3-Pacific Blue 206D Treg transcript BioLegend Table 2. Simple, robust staining protocol The CellTrace CFSE Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. Staining was quenched by . Data were collected on FACS Canto II (BD) and were analyzed with FlowJo software (TreeStar). Dot plot SSC-H VS FL1-H of the CFSE staining tube, highlighted the percentage of staining. In particular, we illustrated how tracking dye fluorescence profiles could be used to ascertain the precursor frequencies of different subsets in . Then, these CFSE labeled CD4 + T cells were seeded in 96 . The advantage of our protocol is given by the irradiation of MSC and the use of multiple doses, which allowed a more accurate dose-dependent estimation of the inhibitory effect. ZERO . Staining Buffer Set (eBiosciences, UK) following the manufacturers protocol, after cell surface marker staining. A carboxyfluorescein diacetate succinimidyl ester (CFSE)-based prolifera-tion assay was used[4]. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. Bioz Stars score: 97/100, based on 1 PubMed citations. It binds to double stranded DNA by intercalating between base pairs in G-C-rich regions. For further details on these alternatives, please refer to the "Limitations" section below. Optimized Multicolor Immunophenotyping Panels Publications from Cytometry Part A - Published, validated resources for developing flow cytometry staining panels! 3.1 CFSE Labeling and Stimulation 1. CellTrace Violet Cell Proliferation Kit | 3 1.4 Quench any unbound dye by adding 5 times the original staining volume of complete culture medium to the cells and incubating them for 5 minutes. Solid line histogram (control) shows unlabeled cells analyzed after 48 hours of cell culture. BioLegend: 352706: Add 5 L to the 20 L of uEVs in PBS: APC anti human TSPAN33 (BAAM) antibody: BioLegend: 395406: An equal volume of fetal bovine serum was added to halt the reaction, and the cells were washed 3 times with cDMEM. BrdU (Bromodeoxyuridine / 5-bromo-2'-deoxyuridine) is an analog of the nucleoside thymidine used in the BrdU assay to identify proliferating cells. 1. Calculate the total number of freshly isolated PBMCs needed for the experiment (3-4 10 6 PBMCs per condition). 3. Freeze/thaw cycles should be avoided ( see Note 4 ). To stain 1 mL of cells in protein-free medium, 1 L of this stock solution is typically used.
Human peripheral blood mononuclear cells were stained with BioLegend's CFSE Cell Division Tracking Kit (Cat # 423801), and then stimulated with (filled histogram) or without (open histogram) PHA for 5 days. CFSE is able to passively diffuse into cells. Cells should be stained for 20 minutes at room temperature with gentle agitation. epitopes.
480017) and MojoSort Magnet (Cat. . Cells were then washed with PBS buffer, re-suspended in stain buffer and analyzed using an ACEA NovoCyte flow cytometer. ZERO BIAS - scores, article reviews, protocol conditions and more. CFSE dilution analysis 1. Similar to CFSE dye, other fluorophores for dye dilution proliferation assay have been developed for encompassing broad range of excitation/emission spectrum . Several different fluorochromes can be used to stain non-viable cells including 7-amino actinomycin D (7-AAD).