GPR43 and GPR109a expression levels on LP CD4 T cells were determined ex vivo and after four days of culture. LP CD4 T cells were cultured in the presence or absence of TCR activation and butyrate (0.5mM). LP CD4 T cell flow staining was determined by CD45 (PerCp-Cy5.5, eBiosciences, Clone: 2d1), CD3 (PE-Dazzle, Biolegend, Clone UCHT1), CD4 (VF450, TONBO, Clone: RPA-T4), CD8 (PE-Dazzle, TONBO, Clone: RPA-T8) and viability dye. Cells were also stained for GPR109a (PE, R&D, clone: 245106) and GPR43 (AF647, Bioss Antibodies, clone: polyclonal) expression. GPR109a expression was also measured on IEMC using the following panel: CD45 (PerCp-Cy5.5; Invitrogen, Clone: 2D1), CD3 (PE-Dazzle, Biolegend, Clone UCHT1), CD19 (PECy5, BD Biosciences, Clone HIB19, HLA-DR (APC-Cy7, BioLegend, Clone: L243) and viability dye. Isotype controls were used to determine positive staining.
Percp cy5
Manufactured by Thermo Fisher Scientific
Sourced in United States
PerCP-Cy5.5 is a fluorescent dye that can be used for flow cytometry applications. It is a tandem dye, combining the properties of PerCP (peridinin-chlorophyll-protein complex) and Cy5.5 (cyanine 5.5). PerCP-Cy5.5 exhibits excitation and emission wavelengths that are compatible with commonly used flow cytometry instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (8 mentions)
Lsm 510 meta, by Zeiss (8 mentions)
Efluor 450, by Thermo Fisher Scientific (6 mentions)
Lsrfortessa, by BD (5 mentions)
Apc cy7, by Thermo Fisher Scientific (5 mentions)
Apc efluor 780, by Thermo Fisher Scientific (4 mentions)
Rb6 8c5, by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (3 mentions)
Facscan flow cytometer, by BD (3 mentions)
Alexa 647, by Thermo Fisher Scientific (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
Apc cy7, by BioLegend (2 mentions)
Apcef780, by Thermo Fisher Scientific (2 mentions)
Efluor 780, by Thermo Fisher Scientific (2 mentions)
Cd62l pe, by BD (2 mentions)
Cd62l pe cy7, by BioLegend (2 mentions)
Cd4 pe fitc apc, by BD (2 mentions)
41bb apc pe, by BD (2 mentions)
Cd8 pe cy7 fitc, by BD (2 mentions)
Ox40 fitc pe cy7, by BD (2 mentions)
62 protocols using percp cy5
1
GPR43 and GPR109a Expression in Gut T Cells
2
Isolation and Characterization of SVF
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The stromal vascular fraction from epididymal fat depots was isolated as described above. After lysing erythrocytes, cells were resuspended in FACS buffer (2% FCS in PBS) and counted on a CASY Counter. Here, 1,000,000 cells were used for cell surface staining, whereas 3,000,000 ones for intracellular staining. Co-cultures were separated using Cell Stripper (ThermoFisher) and resuspended in FACS buffer. Cell surface antigens were blocked with Anti-Mouse CD16/CD32 (1/300,14-0161, eBioscience) and stained for TCRb (1/400, PerCpCy5.5, 45-5961 Invitrogen), TCRgd (1/400, APC, 17-5711 Invitrogen), CD4 (1/400, PeCy7, 25-0041, Invitrogen), CD8 (1/400, APC-eFluor 780, 47-0081 Invitrogen), CD11c (1/400, PerCpCy5.5 45-0114 Invitrogen), CD19 (1/400, PeCy7 25-0193 Invitrogen), CD11b (1/400, PerCpCy5.5 45-0112 Invitrogen), CD11b (1/400, APC 17-0112 Invitrogen), CD11b (1/400, AF488, 53-0112-82, Invitrogen) F4/80 (1/400, PeCy7 25-4801), F4/80 (1/400, AF700, 56-4801-82 Invitrogen), and CD206 (1/400, FITC 123005 Biolegend). Staining of Tregs was done using Mouse Regulatory T Cell Staining Kit (88-8111 eBioscience). FACS measurement was done on a Canto II or LSR II (BD Biosciences) and FlowJo was used for analysis.
3
Multiparameter Flow Cytometry Analysis
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Draining lymph nodes were collected, and single-cell suspensions were prepared and divided into two FAC tubes, to be stained for two different panels (one with surface markers for T cells and one for APCs). Timings for lymph node collection are detailed in the figure legends and the Results section. Briefly, cells were first stained with the viability dye (eF506, eBioscience), blocked for non-specific FcR binding, and then incubated with a master mix of the fluorochrome-conjugated antibodies for 30 min, at 4°C. The markers on the T-cell panel were CD4 (FITC, Invitrogen), CD44 (PerCP Cy5.5, Invitrogen), CD62L (e450, Invitrogen), and ICOS (PeCy7, Biolegend). The APC panel comprised: CD19 (PerCP Cy5.5, Invitrogen), CD11c (e450, Invitrogen), MHC II (H-2b; BV786, Biolegend), CD80 (FITC, BD Biosciences), and CD86 (PeCy7, Biolegend). Data were acquired on either BD LSRFortessa or BD LSR II (BD Biosciences) and analysed using FlowJo 10 software (Tree Star).
4
Multiparameter Flow Cytometry Assay for Immune Cell Profiling
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We used the following antibodies and reagents: anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5, or -APC, anti-CD8 (clone: 53-6.7)-FITC, -PerCP-Cy5, or -APC, anti-IFN-γ (clone: XMG1.2)-APC, anti-CD44 (clone: IM7)-APC, anti-BrdU (clone: Bu20a)-PE, 7-AAD, anti-IL12 (clone: C15.6)-PE, anti-IL4 (clone: 11B11)-PE, anti-IL22 (clone: Poly5164)-PE, anti-IL10 (clone: JES5-16E3)-PE, anti-IL9 (clone: MH9A4)-PE, and anti-CD69 (clone: H1.2F3)-PE (all from eBioscience); anti-caspase-3 (clone: C92605)-FITC (From BD Biosciences); anti-FasL (SC6237) (Santa Cruz Biotechnology); and IgG-Fab2 (clone: 4412) (Cell Signaling).
5
Peripheral Blood Lymphocyte Immunophenotyping
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Anti-Mouse CD8a and Anti-Mouse CD4, labelled with phycoerythrin (PE) and PerCP-Cy5.5, respectively, were purchased from eBioscience (San Diego, CA, USA) Each test contained 50 µL of peripheral blood sample, and then incubated 0.25 µL PE labeled Anti-Mouse CD8a and 0.25 µL PerCP-Cy5.5 labeled Anti-Mouse CD4. After incubating for 20 min in the dark, 2 mL lysing solution (Becton Dickinson, Franklin Lakes, NJ, USA, 10%, v/v) was added to make the lysis of erythrocytes more complete and surviving cells were washed twice with PBS, with centrifugation between each step for 5 min at 500 g. FACScan flow cytometer was used for FCM analysis (BD Biosciences, San Jose, CA, USA). First, an electronic gate was set on lymphocytes and then gated CD4+ T cells in Y axis and CD8+ cells as figure. Dead cells were excluded according to the forward scatter and side scatter.
6
Multiparametric Flow Cytometry Analysis
7
Multiparametric Flow Cytometry Analysis
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For analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA. Intracellular staining of the transcription factors Foxp3 was performed using the Foxp3 Fix/Perm Buffer Set (Biolegend). For detection of intracellular cytokines, cells were first stimulated for 4 h with 50ng/ml PMA and 1 µg/ml ionomycin in the presence of Brefeldin A (All obtained from Sigma), followed by staining for surface markers. Cells were then fixed and permeabilized using the Foxp3 Fix/Perm Buffer Set (Biolegend) and stained for intracellular cytokines. The following antibodies were used at a dilution of 1/200–1/600: PerCP-Cy5.5, PE-, FITC- or APC-labeled anti-IL-17 (TC11-18H10.1), PE- or APC-labeled anti-IL-10 (JES5-16E3), PE-labeled anti-Foxp3 (FJK-16s, eBioscience), PE-, FITC- or APC-labeled anti-CCR6 (29-2L17), PE-, FITC- or APC-labeled anti-CD4 (RM4-5), PE-Cy7-labeled anti-CD3 (145-2C11), APC- or PE-Cy7-labeled anti-IFN (XMG1.2), FITC-, PerCP-Cy5.5 or Pacific Blue-labeled anti-CD45 (30-F11), PE-anti-CCR4 (2G12), PE- or FITC-labeled anti-CCR9 (CW-1.2). All antibodies were obtained from Biolegend unless otherwise noted. Flow cytometry data were acquired on a 5-color FACScan (Becton Dickinson), and analyzed using FlowJo software (Treestar). Cell sorting was performed using a FACSAria II.
8
Quantifying Infiltrated Neutrophils in Peritoneal Lavage
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To determine the number of infiltrated neutrophils in the peritoneal lavage, the cells were first fixed with Rotihistofix (Roth) at 4 °C for 30 min and washed several times in PBS + 3% FBS. Cells were labeled with Gr-1-FITC (1.5 μL RB6–8C5, Thermo Fisher Scientific, 11–5931–82) and F4/80 PerCP-Cy5.5 (1.5 μL BM8, Thermo Fisher Scientific, 45–4801–82) for at least 30 min on ice, washed several times, and followed with the flow cytometer CyFlow Space (Partec) using FloMax software (v. 2.70, Quantum Analysis group), where the FL1 and FL3 channels were used to discriminate between different cell populations (neutrophils and macrophages). The number of cells/ml was determined and the total number of Gr-1+, F4/80− cells was calculated taking into account the sample volume and the percentage of cells in the defined region. Example plots can be found in Supplementary Fig. 25 .
9
Single-cell Tetramer Staining Protocol
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Single-cell suspensions were stained with PE or APC-labelled IAb/NP311-325 or APC-labelled Db/NP368-374 tetramers (NIH tetramer core) at 37°C, 5% CO2 for 2 h in complete RPMI (RPMI with 10% foetal calf serum, 100 μg/ml penicillin-streptomycin, and 2 mM l -glutamine) containing Fc block (24G2). Surface antibodies were added and the cells were incubated for a further 20 min at 4°C. Antibodies used were: anti-CD3 BV785 (BioLegend; clone: 17A2), anti-CD4 BUV805 (BD Bioscience; clone: RM4-5), anti-CD8 BV421 (ThermoFisher; clone: 53-6.7), anti-CD44 BUV395 (BD; clone: IM7), anti-CD45.2 BV605 (BioLegend; clone: 104), anti-CD69 PE-Cy7 (ThermoFisher; clone: H1.2F3), anti-CD127 APC (ThermoFisher; clone: A7R34), anti- γδ TCR PE-Cy7 (BioLegend; clone: GL3), anti-ICOS BV785 (BioLegend; clone: C398.4A), anti-NK1.1 APC-Cy7 (BioLegend; clone: PK136), anti-PD1 BV711 (BioLegend; clone: 29F,1A12), and ‘dump’ antibodies: B220 (RA3-6B2), F4/80 (BM8), and MHC II (M5114) all on eFluor-450 (ThermoFisher) or PerCP-Cy5.5 (ThermoFisher; B220 and F4/80, and BioLegend; MHCII). Cells were stained with a fixable viability dye eFluor 506 (ThermoFisher). Stained cells were acquired on a BD LSR Fortessa and analysed using FlowJo (version 10, BD Bioscience).
10
Comprehensive Antibody Panel for Flow Cytometry
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Example 6
Antibodies that were purchased from BD Biosciences are: APC conjugated anti-CD31 (MEC 13.3), v500 conjugated anti-CD4 (RM4-5), FITC or PE-CF594 conjugated Mouse anti-CD45 (104 or 30-F11), purified Hamster anti-CD3e (500A2), Ly6C-BV605 (AL-21). Antibodies that were purchased from Invitrogen are: purified rabbit anti-GFP (A-11122) and FITC-conjugated anti-rabbit polyclonal. Antibodies that were purchased from eBioscience are: PE conjugated anti-CD8 (CT-CD8b), PE conjugated anti-CD3e (145-2C11), eF450 conjugated anti-CD25 (PC61.5), APC conjugated anti-CD11b (M1/70), Biotin conjugated anti-CD11c (N418), F4/80-Pecy7 (BM8). Antibodies that were purchased from Invitrogen MP are: Alexa 647, Alexa 488, or PercpCy5.5-conjugated streptavidins. Antibodies that were purchased from Jackson Lab are: Cy3 conjugated anti-Syrian Hamster. Antibodies that were purchased from Biolegend are: CD206-PE (C068C2). gp38 serum was a gift of A. Farr.
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