ROR gamma (t) Antibody PE 12-6988-82 from Thermo Fisher Scientific

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Nature communications
July 22 2021

Fig 1: Gene profiling reveals tissue-specific transcriptomic signature of ?dT17 cells.a Left panel, principal component analysis (PCA) plot of the RNA-seq of Tfam-sufficient splenic (Sp), small intestinal (SI), and large intestinal (LI) ?dT17 cells from 3-week-old (n = 3 for each group) mice. ?dT17 cells of each sample were sorted from an individual mouse. Right panel, Euclidean distance comparison of the transcriptome of each splenic ?dT17 cell sample and the mean intestine centroid vs. the mean small intestine centroid to each large intestinal ?dT17 cell sample (n = 3 for each group) (***P = 0.0002). b Heatmap of tissue-specific genes (TSGs) identified by the RNA-seq in Sp, SI, and LI ?dT17 cells. Genes were ranked in a descending order based on the fold changes of expression (Sp/[SI and LI average mean]; [SI and LI average mean]/Sp). c RNA-seq FPKM values of Ahr direct target genes of Sp, SI, and LI ?dT17 cells (mean ± SD) (n = 3 for each group) (Sp vs. SI: Ahr, ***P = 0.0005, Ahrr, **P = 0.0037, Asb2, ***P = 0.0004, Cd93, ****P < 0.0001, Cd160, ***P = 0.0002, Gem, **P = 0.0096, Hic1, ***P = 0.0002, Il17a, ****P < 0.0001, Il22, *P = 0.0180, Kit, ***P = 0.0004, Nuerl3, ****P < 0.0001, Osgin1, ***P = 0.0002, Vps37b, ****P < 0.0001; Sp vs. LI: Ahr, *P = 0.0460, Ahrr, P = 0.0506, Asb2, **P = 0.0076, Cd93, ****P < 0.0001, Cd160, ***P = 0.0001, Gem, *P = 0.0108, Hic1, ***P = 0.0008, Il17a, **P = 0.0071, Il22, *P = 0.0171, Kit, *P = 0.0166, Nuerl3, *P = 0.0137, Osgin1, **P = 0.0027, Vps37b, **P = 0.0034). FPKM, fragments per kilobase of exon model per million mapped fragments. d Ahr and c-Kit expression in Sp, SI, and LI ?dT17 cells by flow cytometry (n = 4 for each group) (Sp vs. SI: Ahr, ***P = 0.0003, c-Kit, **P = 0.0049; Sp vs. LI: Ahr, ****P < 0.0001, c-Kit, **P = 0.0039). Compiled data from one experiment. e Percentages of IL-22+ cells in Sp, SI, and LI ?dT17 cells by flow cytometry (n = 4 for each group) (Sp vs. SI: PMA + Iono, **P = 0.0046, IL-1ß + IL-23, ****P < 0.0001; Sp vs. LI: PMA + Iono, **P = 0.0021, IL-1ß + IL-23, **P = 0.0027). Cells were stimulated with or without PMA (20 ng/ml) plus ionomycin (Iono) (1 µg/ml) but with IL-1ß (10 ng/ml) plus IL-23 (10 ng/ml) for 6 h. Compiled data from one experiment. f GO analysis of the 113 TSGs in ?dT17 cells. g Pie graph of differentially expressed genes (DEGs) between Ctr and Tfam-deficient ?dT17 cells from Sp, SI, and LI. h Heatmap of the 35 core Tfam-regulated DEGs in ?dT17 cells. KO indicated Tfamfl/flRorc-cre mice. i Left panel, PLZF expression in splenic ?dT17 cells from mice with indicated genotypes by flow cytometry (n = 5 for each group) (splenic, ****P < 0.0001; gut, ***P = 0.0008). Compiled data from two independent experiments. Right panel, PLZF expression in gut ?dT17 cells from mice with indicated genotypes by flow cytometry (n = 3 for each group). Compiled data from one experiment. j Left panel, Ki-67 expression in splenic ?dT17 cells from 3-week-old mice with indicated genotypes by flow cytometry (n = 5 for each group) (spleen, ***P = 0.0001; gut, ***P = 0.0003). Compiled data from two independent experiments. Right panel, Ki-67 expression in gut ?dT17 cells from 1-week-old mice with indicated genotypes by flow cytometry (n = 4 for each group). Compiled data from one experiment. Ctr included Tfamfl/fl, Tfam+/+Rorc-cre, and Tfamfl/+Rorc-cre mice in g–j. Data are shown as mean ± SD in a, c–e, i, j.
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Nature communications
July 22 2021

Fig 2: ROR?t-specific ablation of Tfam leads to small intestine tissue remodeling with enhanced tuft cell–ILC2 circuit.Ctr included Tfam+/+Rorc-cre and Tfamfl/+Rorc-cre mice in a, b, f. KO indicated Tfamfl/flRorc-cre mice. a Differential gene expression by RNA-seq analysis of small intestine tissues of Ctr (n = 3) and KO mice (n = 3) at 12-week-old age (fold change =1.5, q value =0.05). b Heatmap of indicated gene expression from RNA-seq analysis of small intestine tissues of Ctr and KO mice. Each sample represented one individual mouse. c Analysis of tuft cell-related gene expression by qRT-PCR in the small intestine tissues of 12-week-old Ctr and Tfamfl/flRorc-cre mice (n = 3) (Dclk1, ****P < 0.0001; Trpm5, ****P < 0.0001; Gnat3, ***P = 0.0002; Plcb2, **P = 0.0023; Il25, **P = 0.0030). Representative data of three independent experiments. Ctr included Tfam+/+, Tfamfl/+, Tfamfl/fl, Tfam+/+Rorc-cre, and Tfamfl/+Rorc-cre mice in c, e, g–o. d Confocal analysis of tuft cells by DCLK1 staining in the small intestine (ileum) of 12-week-old mice with indicated genotypes. Red, DCLK1 staining. Blue, DAPI nuclear staining. Scale bar, 200 µm. Representative data from three independent experiments. e Quantification of tuft cells (DCLK1+) in each small intestine villus viewed by confocal microscopy (n = 9 villi for each group) (****P < 0.0001). Compiled data from two independent experiments. f Heatmap of indicated gene expression from RNA-seq analysis of small intestine tissues of Ctr and KO mice. g Analysis of Il4, Il5, Il13, and Areg expression by qRT-PCR in small intestine tissues of 12-week-old Ctr and Tfamfl/flRorc-cre mice (n = 3) (Il4, ***P = 0.0001; Il5, ****P < 0.0001; Il13, **P = 0.0066; Areg, **P = 0.0023). Representative data of three independent experiments. h IL-4, IL-5, and IL-13 expression in ILC2s or CD4+ T cells by flow cytometry from the small intestine draining lymph nodes (siLN) of Ctr (n = 5) and Tfamfl/flRorc-cre (n = 4) mice at 12-week-old age (ILC2: IL-4, **P = 0.0082; IL-5, **P = 0.0064; IL-13, **P = 0.0097; CD4+ T cells: IL-4, P = 0.0929; IL-5, P = 0.0652; IL-13, *P = 0.0194). Compiled data from two independent experiments. i Confocal analysis of ILC2s (CD3–KLRG1+) in the small intestine (ileum) of 12-week-old mice with indicated genotypes. Red, CD3 staining. Green, KLRG1 staining. White, EpCAM staining. Scale bar, 200 µm. Representative data of three independent experiments. j Quantification of ILC2s (CD3–KLRG1+) in each small intestine villus viewed by confocal microscopy (****P < 0.0001). Ctr, n = 21, Tfamfl/flRorc-cre, n = 22. Compiled data from two independent experiments. k Small intestine length in Ctr or Tfamfl/flRorc-cre mice treated with anti-Thy1.2 neutralizing antibody for 3 weeks (n = 3 for each group) (Ctr vs. Tfamfl/flRorc-cre, **P = 0.0098; Tfamfl/flRorc-cre vs. Tfamfl/flRorc-cre + anti-Thy1.2, *P = 0.0312). Compiled data from two independent experiments. l Picture of small intestines in mice of indicated genotypes treated with anti-IL-4 antibody (100 µg/mouse) or anti-IL-13 antibody (50 µg/mouse) twice a week from 6-week-old age for 3 weeks by intraperitoneal cavity injection. Representative data of two independent experiments. m Small intestine length. Compiled data from two independent experiments (Ctr vs. Tfamfl/flRorc-cre, ****P < 0.0001; Ctr vs. Tfamfl/flRorc-cre + anti-IL-4, ***P = 0.0009; Tfamfl/flRorc-cre vs. Tfamfl/flRorc-cre + anti-IL-4, ***P = 0.0006; Tfamfl/flRorc-cre vs. Tfamfl/flRorc-cre + anti-IL-13, ****P < 0.0001; Tfamfl/flRorc-cre + anti-IL-4 vs. Tfamfl/flRorc-cre + anti-IL-13, **P = 0.0022). n = 5 for Ctr, n = 4 for Tfamfl/flRorc-cre mice; n = 3 for Tfamfl/flRorc-cre mice treated with anti-IL-4; n = 4 for Tfamfl/flRorc-cre mice treated with anti-IL-13. n Analysis of Relmb expression by qRT-PCR in the small intestine tissues of 12-week-old Ctr and Tfamfl/flRorc-cre mice (n = 3) mice at 14 days after Hpb infection (****P < 0.0001). Representative data of three independent experiments. o Left panel, Hpb worm burden in small intestine lumen of Ctr (n = 8) and Tfamfl/flRorc-cre mice (n = 7) at 14 days after Hpb infection (****P < 0.0001). Right panel, Hpb egg counts per gram feces in Ctr (n = 8) and Tfamfl/flRorc-cre mice (n = 7) mice at 14 days after Hpb infection (***P = 0.0008). Compiled data from three independent experiments. Data are shown as mean ± SD in c, e, g, h, j, k, m–o. *, **, ***, **** indicated P values =0.05, 0.01, 0.001, 0.0001, respectively.
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Nature communications
July 22 2021

Fig 3: Tfam deletion in ROR?t+ lymphocytes lead to global metabolomic changes in the small intestine.Ctr included Tfam+/+Rorc-cre and Tfamfl/+Rorc-cre mice, while KO indicated Tfamfl/flRorc-cre mice in a–e. a PCA analysis of the metabolome of small intestine tissues of 12-week-old Ctr and KO mice (n = 3 for each group). Each group included three individual mice. b Heatmap of the differentially regulated metabolites between small intestine tissues of Ctr and KO groups identified by the metabolome analysis. Metabolites were ranked in a descending order based on the fold changes of abundance (Ctr/KO; KO/Ctr). c Heatmap of indicated gene expression of small intestine tissue RNA-seq of Ctr and KO mice as shown in Fig. 5a. Genes were ranked in a descending order based on the fold changes of expression (Ctr/KO). d RNA-seq FPKM values of indicated genes in small intestine tissues of the mice (n = 3) (Fabp6, *P = 0.0155; Ugt1a1, **P = 0.0019; Slc10a2, *P = 0.0162; Sult1d1, *P = 0.0158; Slc51b, **P = 0.0027; Slc51a, **P = 0.0066; Gsr, *P = 0.0186). e Heatmap of DEGs in small intestine tissues of the mice identified by RNA-seq (n = 3). Genes were ranked in a descending order based on the fold changes of expression (KO/Ctr). Ctr included Tfam+/+, Tfamfl/+, Tfamfl/fl, Tfam+/+Rorc-cre, and Tfamfl/+Rorc-cre mice in g–k. f Confocal analysis of hypoxia by pimonidazole staining in the small intestine (ileum) of 12-week-old mice with indicated genotypes. Red, pimonidazole staining. Blue, DAPI staining. Scale bar, 200 µm. Representative data of three independent experiments. g Pimonidazole MFI in the epithelial cells shown in f (n = 5 for each group) (***P = 0.0006). Compiled data from one experiment. h Bodipy uptake in small intestine epithelial cells from Ctr and Tfamfl/flRorc-cre mice measured by flow cytometry. Representative data of two independent experiments. i Bodipy MFI in the epithelial cells as shown in h (n = 3 for each group) (***P = 0.0006). Compiled data from one experiment. j Body weight of 5-month-old female Ctr (n = 3) and Tfamfl/flRorc-cre mice (n = 5) and male Ctr (n = 4) and Tfamfl/flRorc-cre mice (n = 3) (female, *P = 0.0169; male, ***P = 0.0006). Compiled data from two independent experiments. k Visceral adipose tissue (VAT) weight (n = 4) and 4-day-food uptake (n = 5) in 5-month-old mice with indicated genotypes (VAT, **P = 0.0039; food uptake, *P = 0.0321). Compiled data from two independent experiments. Data are shown as mean ± SD in d, g, i–k.
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Nature communications
July 22 2021

Fig 4: Tfam deficiency in ?dT17 cells cause small intestine lengthening.a Picture of the small intestine of 8-week-old littermate Tfam+/+ Rorc-cre and Tfamfl/flRorc-cre mice. Representative data of three independent experiments. b Small intestine length of mice with indicated genotypes at different ages (week 3, P = 0.8542; week 6, P = 0.056; week 8, *P = 0.03853; week 12, ****P < 0.0001). Ctr, n = 5 for 3-week-old and 12-week-old age, n = 4 for 6-week-old age, n = 3 for 8-week-old age, Tfamfl/flRorc-cre, n = 4. Compiled data from three independent experiments. c Hematoxylin and eosin (H&E) staining of the small intestine from 12-week-old mice with indicated genotypes. Representative data of two independent experiments. Scale bar, 100 µm. d Histology score of the small intestine (n = 3 for each group) (*P = 0.0135). Each dot represented the data from an individual mouse. Compiled data from one experiment. e ROR?t and CD45.1 expression in Lin– cells (upper panel) or total ?dT cells (lower panel) measured by flow cytometry in Ctr or Tfamfl/flRorc-cre mice with or without CD45.1+ wild-type (WT) bone marrow (BM) transfer. Representative data of two independent experiments. f Percentages of ?dT17 cells in total ?dT cells (Ctr vs. Tfamfl/flRorc-cre, ****P < 0.0001; Ctr + CD45.1+ BM vs. Tfamfl/flRorc-cre + CD45.1+ BM transfer, ***P = 0.0002). g Percentages of ILC3s in Lin– cells (Ctr vs. Tfamfl/flRorc-cre, ****P < 0.0001; Ctr + CD45.1+ BM vs. Tfamfl/flRorc-cre + CD45.1+ BM, *P = 0.0382; Tfamfl/flRorc-cre vs. Tfamfl/flRorc-cre + CD45.1+ BM, **P = 0.0059). h Percentages of CD45.1+ ILC3s in total ILC3s (Ctr + CD45.1+ BM vs. Tfamfl/flRorc-cre + CD45.1+ BM, ***P = 0.0002). i Picture of the small intestine. j Small intestine length. Compiled data from two independent experiments in f–h, j (Ctr vs. Tfamfl/flRorc-cre, **P = 0.0037; Ctr + CD45.1+ BM vs. Tfamfl/flRorc-cre + CD45.1+ BM, **P = 0.0014) (n = 4 for Ctr with or without BM transfer groups and n = 5 for Tfamfl/flRorc-cre mice with or without BM transfer groups). k Picture of the small intestine of mice with indicated genotypes. Five-week-old age mice were treated with tamoxifen (2 mg/mouse by intraperitoneal injection) daily for 5 days. Data were collected 3 weeks after the last tamoxifen injection. l Small intestine length. Compiled data from three independent experiments (n = 6 for Ctr, n = 7 for Tfamfl/flTcrdCreER mice). Ctr included Tfam+/+, Tfamfl/+, Tfamfl/fl, Tfam+/+Rorc-cre, and Tfamfl/+Rorc-cre mice in b, d, f–h, j (**P = 0.0060). Ctr included Tfam+/+TcrdCreER and Tfamfl/+TcrdCreER mice in l. Data are shown as mean ± SD in b, d, f–h, j, l.
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Nature communications
July 22 2021

Fig 5: Tfam is critical for ?dT17 cell and ILC3 maintenance.a Flow cytometry analyses of ILC3s (upper panel, gated on lineage-negative lymphocytes) and ?dT17 cells (lower panel, gated on ?dT cells) from small intestine lamina propria (LPL) of 3-week-old mice of indicated genotypes. Representative data of three independent experiments. Lineage markers in b and c include CD3, CD5, CD19, Ly6G, CD11b, and CD11c. b ?dT17 cell percentages in total ?dT cells (percentage, *P = 0.0120; cell number, *P = 0.0106), and ILC3 percentages in lineage-negative (Lin–) cells (upper panel) (P = 0.1575) and cell numbers (lower panel) (P = 0.1725) from the small intestine of mice shown in a (n = 4 for each group). Compiled data from two independent experiments. c ?dT17 cell percentages in total ?dT cells and ILC3 percentage in lineage-negative cells (upper panel) (?dT17: week 3, ***P = 0.0002, week 6, ****P < 0.0001; ILC3: week 6, **P = 0.0019) and cell numbers (lower panel) (?dT17: week 3, **P = 0.0035, week 6, *P = 0.0101; ILC3: week 6, *P = 0.0396) of the large intestine of mice at indicated ages (n = 4 for each group). The rest time points showed no significance between Ctr and Tfamfl/flRorc-cre (P > 0.05). Compiled data from two independent experiments. d Th17 cell percentages in total CD4+ T cells (upper panel) (P = 0.1705) and cell numbers (lower panel) (P = 0.0950) of the large intestine of 6-week-old age mice (n = 4 for each group). Compiled data from two independent experiments. e ROR?t expression in ?dT cells of various organs (Sp spleen, pLN peripheral lymph nodes) of mice with indicated genotypes measured by flow cytometry. Representative data of three independent experiments. f ?dT17 cell percentages in total ?dT cells from the various organs as shown in e (n = 4 for each group). Sp, *P = 0.0121; pLN, ****P < 0.0001; liver, **P = 0.0043; lung, ***P = 0.0009; fat, ****P < 0.0001; skin, ****P < 0.0001. Compiled data from two independent experiments. Ctr included Tfam+/+, Tfamfl/+, Tfamfl/fl, Tfam+/+Rorc-cre, and Tfamfl/+Rorc-cre mice. Data are shown as mean ± SD in b–d, f.

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Thermo Fisher Scientific for
ROR gamma (t) Antibody PE

The ROR gamma (t) Antibody PE from Thermo Fisher Scientific is a Rat Monoclonal antibody to RORC. This antibody recognizes Homo Sapiens, Human, Mouse, and Non-Human Primate antigen. The ROR gamma (t) Antibody PE has been validated for the following applications: Flow Cytometry.

Product Specs
ItemROR gamma (t) Antibody PE
CompanyThermo Fisher Scientific
Price Pricing Info Supplier Page View Company Product Page
Catalog Number12-6988-82
Quantity100 µg
ApplicationsFlow Cytometry
Concentration0.2 mg/mL
Conjugate/TagPE
FormatPBS with 0.09% sodium azide; pH 7.2
ReactivityHuman, Mouse, Rhesus Monkey
TargetROR gamma (t)
IsotypeIgG2a kappa
CloneAFKJS-9
HostRat
NCBI Full Gene NameRAR related orphan receptor C, RAR-related orphan receptor gamma
NCBI Gene AliasesRZRG, IMD42, Nr1f3, TOR, Thor, RORgamma, NR1F3, RORG, RZR-GAMMA
ClonalityMonoclonal
PurityAffinity chromatography
Storage Temperature4° C, store in dark, DO NOT FREEZE!
Antibody TypePrimary
Storage4° C, store in dark, DO NOT FREEZE!

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(15) Characterization of the pathoimmunology of necrotizing enterocolitis reveals novel therapeutic opportunitiesNature communicationsNovember 13, 2020Cho SX, Rudloff I, Lao JC, Pang MA, Goldberg R, Bui CB, McLean CA, Stock M, Klassert TE, Slevogt H, et al.(Biolegend, cat# 653814, 1:25), T-bet-PE-CF594 (BD, cat# 562467, 1:25) and RORγt-PE (eBioscience, cat# 12-6988-82, 1:200) was performed in permeabilization buffer (eBioscience) for 30 min at 4 °C. At least 300,000

(16) IL-6, NLR, and SII Markers and Their Relation with Alterations in CD8+ T-Lymphocyte Subpopulations in Patients Treated for Lung AdenocarcinomaBiologyNovember 5, 2020Islas-Vazquez L, Aguilar-Cazares D, Galicia-Velasco M, Rumbo-Nava U, Meneses-Flores M, Luna-Rivero C, Lopez-Gonzalez JS.

(17) Insulin-Like Growth Factors Are Key Regulators of T Helper 17 Regulatory T Cell Balance in AutoimmunityImmunityApril 14, 2020DiToro D, Harbour SN, Bando JK, Benavides G, Witte S, Laufer VA, Moseley C, Singer JR, Frey B, Turner H, et al.1A5,Cell Signaling,9206S P70 S6K T421/S424 9204,Cell Signaling,9204S RORγt PE AFKJS-9,eBioscience,12-6988-82 RORγt APC AFKJS-9,eBioscience,17-6988-82 S6 S235/236 Pacific Blue D57.2.2E,Cell Signaling,8520S Streptavidin

(18) Neurokinin-1 Receptor Signaling Is Required for Efficient Ca²⁺ Flux in T-Cell-Receptor-Activated T CellsCell reportsMarch 10, 2020Morelli AE, Sumpter TL, Rojas-Canales DM, Bandyopadhyay M, Chen Z, Tkacheva O, Shufesky WJ, Wallace CT, Watkins SC, Berger A, et al.L50-820), PE",BD,Cat # 560074 "Anti-mouse RoRγt (Clone AFJKS-9), PE",Invitrogen / eBioscience,Cat # 12-6988-82 "Anti-mouse FoxP3 (Clone FJK-16 s), PE",Invitrogen / eBioscience,Cat # 12-5773-82 Anti-mouse / human

(19) Actin-Binding Protein Cortactin Promotes Pathogenesis of Experimental Autoimmune Encephalomyelitis by Supporting Leukocyte Infiltration into the Central Nervous SystemThe Journal of neuroscience : the official journal of the Society for NeuroscienceFebruary 12, 2020Samus M, Li YT, Sorokin L, Rottner K, Vestweber D.anti-Ly6G, FITC-conjugated rat antiCD11b, APC-conjugated rat anti-CD45, anti-T-bet (#644804), antiROR␥t (#12-6988-82, Invitrogen), anti-IL-17A (#506910), IFN-␥ (#505826), and anti-ICAM1 (YNI-1.1). FITC-conjugated rat

(20) Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3⁺ Regulatory T CellsCell reportsFebruary 4, 2020Yang YH, Istomine R, Alvarez F, Al-Aubodah TA, Shi XQ, Takano T, Thornton AM, Shevach EM, Zhang J, Piccirillo CA.

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(1)

3/31/2023Poor Signal with ROR Gamma (t) Antibody, No Distinct Positive Population Owen JiangI'm interested in studying how diets and dietary metabolites affect host immune response, with a focus of CD4 T cells.
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4/22/2022Good Staining of ILC3s Karl WagnerWe are interested in using cutting edge single cell analysis methods (spectral flow cytometry, scRNAseq, etc) to study the mechanisms responsible for establishing and maintaining immune tolerance.

Nature communications

Nature communications

Nature communications

Nature communications

Nature communications

ROR gamma (t) Antibody PE from Thermo Fisher Scientific

Thermo Fisher Scientific

Citations (113)

Reviews

ROR gamma (t) Antibody PE from Thermo Fisher Scientific

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