Aria 2 cytometer, by BD - Product details

1

Analyzing T Cell Suppression by Flow Cytometry

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PBMCs were incubated for 30 min at 4 °C with specific, labeled monoclonal antibodies, washed, and then sorted using an ARIA II cytometer (BD Biosciences). Naïve T effector cells (Teff) were defined as CD3⁺CD4⁺CD25⁻CD127⁺CD45RA⁺ T cells, Tregs were defined as CD3⁺CD4⁺CD25⁺CD127⁻ T cells, and dendritic cells (DCs) were defined as CD3-CD4-CD14-CD16-CD11c⁺ cells. After cell sorting, Teff was washed and stained with CellTrace CFSE. Next, the cells were washed, incubated with DCs (Teff:DC ratio 1:0.4), Tregs (Teff:Treg ratio 1:0.5) and stimulated by Staphylococcus enterotoxin E (0.2 ng/ml). The cells were cultured for 5 days in complete Panserin medium (Dutscher) in 96-well plates and the proportion of proliferating cells was measured with a MACSquant system (Miltenyi). The data were analyzed with FlowJo software. The percentage of suppression was calculated with the following formula: [log2(y) of (Teff cells alone) – log2(y) of (Teff + Treg cells])/log2(y) of (Teff cells alone) × 100. The y value corresponds to the mean fluorescence intensity of the CFSE of the whole Teff cell population, divided by the mean fluorescence intensity of the CFSE of undivided Teff cells^{40 (link)}.

Hadjadj J., Castro C.N., Tusseau M., Stolzenberg M.C., Mazerolles F., Aladjidi N., Armstrong M., Ashrafian H., Cutcutache I., Ebetsberger-Dachs G., Elliott K.S., Durieu I., Fabien N., Fusaro M., Heeg M., Schmitt Y., Bras M., Knight J.C., Lega J.C., Lesca G., Mathieu A.L., Moreews M., Moreira B., Nosbaum A., Page M., Picard C., Ronan Leahy T., Rouvet I., Ryan E., Sanlaville D., Schwarz K., Skelton A., Viallard J.F., Viel S., Villard M., Callebaut I., Picard C., Walzer T., Ehl S., Fischer A., Neven B., Belot A, & Rieux-Laucat F. (2020). Early-onset autoimmunity associated with SOCS1 haploinsufficiency. Nature Communications, 11, 5341.

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2

Multiparametric Analysis of Immune Cell Populations

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Single cell suspensions were prepared and stained with indicated surface or intracellular antibodies (BD Biosciences, eBioscience, BioLegend, Tonbo, and R&D Systems), as previously described (Adams et al., 2019 (link)). Apoptosis was evaluated by caspase activity staining using the FLICA poly caspase assay kit (Immunochemistry Technologies). NK cell proliferation was analyzed by labeling cells with 5 μM Cell Trace Violet (CTV, Invitrogen) prior to transfer and infection. Flow cytometry was performed on an LSR II (BD). Cell sorting was performed on an Aria II cytometer (BD). All data were analyzed with FlowJo software (TreeStar). Flow cytometry of purified lymphocytes was performed using the following fluorophore-conjugated antibodies: CD3ε (17A2), TCRβ (H57–597), CD19 (ID3), F4/80 (BM8.1), NK1.1 (PK136), Ly49H (3D10), CD45.1 (A20), CD45.2 (104), CD11b (M1/70), CD27 (LG.3A10), KLRG1 (2F1), Ly49D (4E5), Ly49A (YE1/48.10.6), Ly49C/I (5E6), CD69 (H1.2F3), Granzyme A (3G8.5), Granzyme B (GB11), IFN-γ (XMG1.2), CD107a (1D4B), and CD49b (DX5).

Wiedemann G.M., Grassmann S., Lau C.M., Rapp M., Villarino A.V., Friedrich C., Gasteiger G., O’Shea J.J, & Sun J.C. (2020). Divergent Role for STAT5 in the Adaptive Responses of Natural Killer Cells. Cell reports, 33(11), 108498.

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3

Characterizing Myeloid and Lymphoid Populations

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Single cell suspensions enriched for leukocytes were characterized according to their myeloid and lymphoid populations by flow cytometry analysis. Myeloid cell staining was performed following the procedure previously described by Thiele-Orberg et al (Thiele Orberg et al., 2017 (link)). Briefly, MO-IMC were characterized as CD45⁺CD3⁻CD11b^hiLy6C^hiLy6G⁻F4/80^−/lowMHC-II^−/low, PMN-IMC as CD45⁺CD3⁻CD11b^hi Ly6C^intLy6G⁺F4/80⁻MHC-II⁻, and Mϕ as CD45⁺CD3⁻CD11b^intLy6C⁻Ly6G⁻F4/80^hi cells. For intracellular cytokine staining, cells were stimulated for 4.5 h with 30 nM phorbol 12-myristate 13-acetate and 1 μM ionomycin (both eBioscience) in presence of Golgistop (BD). Cells were subsequently stained for cell surface markers (CD4-PerCp/Cy5.5, GK1.5; CD3-AF700, 17A2; CD8-PE/CF594, 53-6.7; all Biolegend) followed by fixation/permeabilization (Cytofix/Cytoperm, BD) and staining using IL-17A-Pacific Blue (TC11-18H10.1, Biolegend). Flow cytometry was performed using a LSRII cytometer (BD) and data were analyzed with FACSDiVa 6.1.3 software (BD). For cell sorting experiments, CEC populations were sorted as CD45-EpCAM+ cells using an AriaII cytometer (BD).

Chung L., Orberg E.T., Geis A.L., Chan J.L., Fu K., DeStefano Shields C.E., Dejea C.M., Fathi P., Chen J., Finard B.B., Tam A.J., McAllister F.M., Fan H., Wu X., Ganguly S., Lebid A., Metz P., Van Meerbeke S.W., Huso D.L., Wick E.C., Pardoll D.M., Wan F., Wu S., Sears C.L, & Housseau F. (2018). BACTEROIDES FRAGILIS TOXIN COORDINATES A PRO-CARCINOGENIC INFLAMMATORY CASCADE VIA TARGETING OF COLONIC EPITHELIAL CELLS. Cell host & microbe, 23(2), 203-214.e5.

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4

Multicolor Flow Cytometry Immunophenotyping

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Single cell suspensions were stained with Live/Dead Yellow (Life Technologies, Grand Island, NY), CD11b-PerCp/Cy5.5 (M1/70, Biolegend, San Diego, CA), I-A/I-E-AF488 (M5/114.15.2, Biolegend), Gr-1-Pacific Orange (RB6-8C5, Life Technologies), F4/80-PE-Cy7 (BM8, Biolegend), CD11c-APC-Cy7 (HL3, BD Biosciences, San Jose, CA), CD117-PE-Cy5 (2B8, Biolegend) and FcεRI (Mar-1, Biolegend). Ly6-C-Pacific Blue (HK1.4, Biolegend) and Ly6-G-AF647 (1A8, Biolegend) were used in adoptive transfer experiments. For intracellular cytokine staining, cells were stimulated for 4.5 h with 30 nM phorbol 12-myristate 13-acetate and 1 μM ionomycin (both eBioscience) in presence of Golgistop (BD). Cells were subsequently stained against cell surface markers (CD4-PerCp/Cy5.5, GK1.5; CD3-AF700, 17A2; CD8-PE/CF594, 53-6.7; all Biolegend) followed by fixation/permeabilization (Cytofix/Cytoperm, BD) and staining using IL-17A-Pacific Blue (TC11-18H10.1, Biolegend). Flow cytometry was performed using a LSRII cytometer (BD) and data were analyzed with FACSDi-Va 6.1.3 software (BD). For cell sorting (FACS) experiments, myeloid populations were sorted using an AriaII cytometer (BD). MO-MDSCs were sorted as CD45⁺CD3^-CD11b^hiGr-1^lowF4/80^-/lowMHC-II^-/low, PMN-MDSCs as CD45⁺CD3^-CD11b^hiGr-1^hiF4/80^-MHC-II^-, and MΦ as CD45⁺CD3^-CD11b^hiGr-1^-/lowF4/80^hiMHC-II⁺ cells.

Orberg E.T., Fan H., Tam A.J., Dejea C.M., Destefano-Shields C.E., Wu S., Chung L., Finard B.B., Wu X., Fathi P., Ganguly S., Fu J., Pardoll D.M., Sears C.L, & Housseau F. (2016). The Myeloid Immune Signature of Enterotoxigenic Bacteroides Fragilis-Induced Murine Colon Tumorigenesis. Mucosal immunology, 10(2), 421-433.

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5

Transcriptional Profiling of Innate Lymphocytes

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BM CLP (lin⁻ c-kit⁺ sca1⁺ flt3⁺ IL-7Ra⁺), ILCP (lin⁻ c-kit⁺ sca1⁺ flt3⁻ IL-7Ra⁺ α4β7⁺), and gut ILC3 (lin⁻ Rorc⁺ IL-7Ra⁺) were sorted to ∼99% purity on an Aria II cytometer (BD). Cell lysis was subsequently performed using Tri-Reagent (Ambion), RNA was purified using the RNeasy kit (with on-column DNase I treatment; QIAGEN), and MuLV reverse transcription and oligo(dT)16 primers (Applied Biosystems) were used for cDNA synthesis. iQ Sybr Green SuperMix (Bio-Rad Laboratories) was used for qRT-PCR. Data were normalized to expression of β-actin and expressed as relative target abundance via the ΔΔCt method, where Ct (threshold cycle) is the cycle number at which the amplification curve intersects the threshold value. The primer sets used for qRT-PCR are the following: Nfil3 forward, 5′-AATTCATTCCGGACGAGAAG-3′; Nfil3 reverse, 5′-CGATCAGCTTGTTCTCCAAA-3′; β-actin forward, 5′-TGCGTGACATCAAAGAGAAG-3′; and β-actin reverse, 5′-CGGATGTCAACGTCACACTT-3′.

Geiger T.L., Abt M.C., Gasteiger G., Firth M.A., O’Connor M.H., Geary C.D., O’Sullivan T.E., van den Brink M.R., Pamer E.G., Hanash A.M, & Sun J.C. (2014). Nfil3 is crucial for development of innate lymphoid cells and host protection against intestinal pathogens. The Journal of Experimental Medicine, 211(9), 1723-1731.

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6

Multiparametric Analysis of Immune Cell Populations

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Single cell suspensions were prepared and stained with indicated surface or intracellular antibodies (BD Biosciences, eBioscience, BioLegend, Tonbo, and R&D Systems), as previously described (Adams et al., 2019 (link)). Apoptosis was evaluated by caspase activity staining using the FLICA poly caspase assay kit (Immunochemistry Technologies). NK cell proliferation was analyzed by labeling cells with 5 μM Cell Trace Violet (CTV, Invitrogen) prior to transfer and infection. Flow cytometry was performed on an LSR II (BD). Cell sorting was performed on an Aria II cytometer (BD). All data were analyzed with FlowJo software (TreeStar). Flow cytometry of purified lymphocytes was performed using the following fluorophore-conjugated antibodies: CD3ε (17A2), TCRβ (H57–597), CD19 (ID3), F4/80 (BM8.1), NK1.1 (PK136), Ly49H (3D10), CD45.1 (A20), CD45.2 (104), CD11b (M1/70), CD27 (LG.3A10), KLRG1 (2F1), Ly49D (4E5), Ly49A (YE1/48.10.6), Ly49C/I (5E6), CD69 (H1.2F3), Granzyme A (3G8.5), Granzyme B (GB11), IFN-γ (XMG1.2), CD107a (1D4B), and CD49b (DX5).

Wiedemann G.M., Grassmann S., Lau C.M., Rapp M., Villarino A.V., Friedrich C., Gasteiger G., O’Shea J.J, & Sun J.C. (2020). Divergent Role for STAT5 in the Adaptive Responses of Natural Killer Cells. Cell reports, 33(11), 108498.

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7

Single Cell Immune Profiling Protocol

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Single cell suspensions were prepared from indicated organs and Fc receptors were blocked with 2.4G2 mAb before staining with the indicated surface or intracellular antibodies (BD, BioLegend, or eBioscience). Flow cytometry was performed on an LSR II (BD). Cell sorting was performed on an Aria II cytometer (BD). All data were analyzed with FlowJo software (TreeStar). NK cell enrichment and adoptive transfers were performed as previously described (9 (link)).

Madera S., Geary C.D., Lau C.M., Pikovskaya O., Reiner S.L, & Sun J.C. (2018). Divergent requirement of T-box transcription factors in effector and memory NK cells. Journal of immunology (Baltimore, Md. : 1950), 200(6), 1977-1981.

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8

Isolation and Characterization of Patellar Tendon Cells

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After dissection of patellar tendons from 5-month-old wildtype and Crtap^-/- mice, tissues were cut into small pieces in 1 × PBS with 10% fetal bovine serum (FBS) and incubated with 500 µl of PBS + 10% FBS and 0.1% collagenase at 37°C for 3 hr. After digestion, cells were filtered with a 40 μm strainer, washed, resuspended in 1 × PBS at a concentration of 10⁶ cells/mL, and stained with CD45-pacific blue (clone: 30-F11), CD31-eFlour 450 (clone: 390), CD146-PE-Cy7 (clone ME-9F1) and CD200-APC (clone OX-90) (eBioscience). Propidium iodide was used for selecting viable cells. Cell analysis was performed using a LSRII Fortessa, and fluorescence-activated cell sorting (FACS) experiments were done using an AriaII cytometer (BD Biosciences, San Jose, CA). Data were analyzed with FlowJo software (TreeStar, OR).

Grol M.W., Haelterman N.A., Lim J., Munivez E.M., Archer M., Hudson D.M., Tufa S.F., Keene D.R., Lei K., Park D., Kuzawa C.D., Ambrose C.G., Eyre D.R, & Lee B.H. (2021). Tendon and motor phenotypes in the Crtap-/- mouse model of recessive osteogenesis imperfecta. eLife, 10, e63488.

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9

Enrichment and Purification of Mouse NK Cells

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NK cells
(TCRβ⁻CD3ε⁻CD19⁻F4/80⁻CD45⁺NK1.1⁺)
were enriched from spleens of pooled C57BL/6 mice by negative selection over
BioMag goat anti-rat IgG beads (Qiagen) coated with rat anti-mouse
CD8α, CD4, CD19, and Ter-119 antibodies (Bio X Cell, clones 2.43,
GK1.5, 1D3, and TER-119 respectively) before being sorted to high purity on
an Aria II cytometer (BD Biosciences).

Adams N.M., Geary C.D., Santosa E.K., Lumaquin D., Le Luduec J.B., Sottile R., van der Ploeg K., Hsu J., Whitlock B.M., Jackson B.T., Weizman O.E., Huse M., Hsu K.C, & Sun J.C. (2019). Cytomegalovirus infection drives avidity selection of natural killer cells. Immunity, 50(6), 1381-1390.e5.

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10

Multicolor Flow Cytometry Immunophenotyping

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Single cell suspensions were stained with Live/Dead Yellow (Life Technologies, Grand Island, NY), CD11b-PerCp/Cy5.5 (M1/70, Biolegend, San Diego, CA), I-A/I-E-AF488 (M5/114.15.2, Biolegend), Gr-1-Pacific Orange (RB6-8C5, Life Technologies), F4/80-PE-Cy7 (BM8, Biolegend), CD11c-APC-Cy7 (HL3, BD Biosciences, San Jose, CA), CD117-PE-Cy5 (2B8, Biolegend) and FcεRI (Mar-1, Biolegend). Ly6-C-Pacific Blue (HK1.4, Biolegend) and Ly6-G-AF647 (1A8, Biolegend) were used in adoptive transfer experiments. For intracellular cytokine staining, cells were stimulated for 4.5 h with 30 nM phorbol 12-myristate 13-acetate and 1 μM ionomycin (both eBioscience) in presence of Golgistop (BD). Cells were subsequently stained against cell surface markers (CD4-PerCp/Cy5.5, GK1.5; CD3-AF700, 17A2; CD8-PE/CF594, 53-6.7; all Biolegend) followed by fixation/permeabilization (Cytofix/Cytoperm, BD) and staining using IL-17A-Pacific Blue (TC11-18H10.1, Biolegend). Flow cytometry was performed using a LSRII cytometer (BD) and data were analyzed with FACSDi-Va 6.1.3 software (BD). For cell sorting (FACS) experiments, myeloid populations were sorted using an AriaII cytometer (BD). MO-MDSCs were sorted as CD45⁺CD3^-CD11b^hiGr-1^lowF4/80^-/lowMHC-II^-/low, PMN-MDSCs as CD45⁺CD3^-CD11b^hiGr-1^hiF4/80^-MHC-II^-, and MΦ as CD45⁺CD3^-CD11b^hiGr-1^-/lowF4/80^hiMHC-II⁺ cells.

Orberg E.T., Fan H., Tam A.J., Dejea C.M., Destefano-Shields C.E., Wu S., Chung L., Finard B.B., Wu X., Fathi P., Ganguly S., Fu J., Pardoll D.M., Sears C.L, & Housseau F. (2016). The Myeloid Immune Signature of Enterotoxigenic Bacteroides Fragilis-Induced Murine Colon Tumorigenesis. Mucosal immunology, 10(2), 421-433.

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Aria 2 cytometer

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15 protocols using aria 2 cytometer

Analyzing T Cell Suppression by Flow Cytometry

Multiparametric Analysis of Immune Cell Populations

Characterizing Myeloid and Lymphoid Populations

Multicolor Flow Cytometry Immunophenotyping

Transcriptional Profiling of Innate Lymphocytes

Multiparametric Analysis of Immune Cell Populations

Single Cell Immune Profiling Protocol

Isolation and Characterization of Patellar Tendon Cells

Enrichment and Purification of Mouse NK Cells

Multicolor Flow Cytometry Immunophenotyping

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