47-0271-82

antibody from Invitrogen Antibodies

Targeting: CD27 S152, TNFRSF7, Tp55

Provider product page for 47-0271-82

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Antibody Data
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Flow cytometry [1]
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Product number: 47-0271-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD27 Monoclonal Antibody (LG.7F9), APC-eFluor™ 780, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The LG.7F9 monoclonal antibody reacts with mouse CD27, a lymphocyte-specific member of the TNFR superfamily. CD27 is expressed by virtually all mature T cells and by a subpopulation of B cells, mainly memory B cells. In mouse, CD27 has been found on nearly all thymocytes excluding a population of CD46-CD8- precursors. CD27 binds to CD70 and, through this interaction, plays an important role in T cell-B cell interaction. It has been reported that triggering CD27 plays an important role in the maturation of CD4+ and CD8+ effector cells. LG.7F9 cross-reacts with human and rat CD27. Applications Reported: This LG.7F9 antibody has been reported for use in flow cytometric analysis. Applications Tested: This LG.7F9 antibody has been tested by flow cytometric analysis of mouse splenocytes. This can be used at less than or equal to 0.25 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. APC-eFluor 780 emits at 780 nm and is excited with the Red laser (633 nm). Please make sure that your instrument is capable of detecting this fluorochome. Light sensitivity: This tandem is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL cell sample + 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 633-647 nm; Emission: 780 nm; Laser: Red Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human, Mouse, Rat
Isotype: IgG
Antibody clone number: LG.7F9
Vial size: 100 µg
Concentration: 0.2 mg/mL
Storage: 4° C, store in dark, DO NOT FREEZE!

CD27 protein structure - 47-0271-82 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

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Experimental details: FIG 1 Lung CD19 + and CD27 + CD19 + cells at day 14 (A and B) and day 28 (C and D) postinfection. C57BL/6 mice were subcutaneously given 30 or 150 mug of anti-CD20 or isotype control antibody weekly. Three days after the first injection, all mice were inoculated with approximately 2 x 10 5 asci of P. murina inoculum. Fourteen or 28 days postinfection, the right upper and lower lung lobes were harvested, and single cells were prepared and stimulated with Pneumocystis (PC) antigen for 6 h followed by staining for cell surface markers and intracellular cytokine staining. For CD19 + and CD27 + CD19 + cells, 100,000 cells were acquired. One-way ANOVA and Tukey's multiple-comparison tests indicated that there were significant differences between anti-CD20 and isotype control antibody (ISO)-treated groups. Statistical significance is indicated as follows: ***, P

Supportive validation

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Experimental details: FIG 6 Lung CD19 + and CD27 + CD19 + cells in secondary PC infection. All mice were inoculated with approximately 2 x 10 5 asci of P. murina inoculum. Six weeks later, the mice were subcutaneously given 30 or 150 mug of anti-CD20 or isotype control antibody weekly. Three days after the first injection, all mice were reinoculated with approximately 2 x 10 5 asci of P. murina inoculum. Fourteen days postinfection, the right upper and lower lung lobes were taken, and single cells were prepared and stimulated with PC antigen for 6 h for surface markers and intracellular cytokine staining. For both CD19 + and CD27 + CD19 + cells, 100,000 cells were acquired. One-way ANOVA and Tukey''s multiple-comparison tests indicated that there were significant differences between anti-CD20- and ISO-treated groups for CD19 + cells but not for CD27 + CD19 + cells. Statistical significance is indicated as follows: **, P

Supportive validation

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Experimental details: Figure 3 Phenotypic characterization of circulating B cell subsets in patients and HC. (A) Gated by CD19 + B cells, IgD + CD27 - NB, IgD + CD27 + USM B cells, IgD - CD27 + SM B cells, and IgD - CD27 - DN B cells were identified. The graphs are representative for HC and patients with different duration. Mean value of each B cell subset's percentage is shown in the quadrants. (B) Statistical graphs for comparison of CD19 + B cells' percentages between patient groups and HC. (C-E) Statistical graphs for distinct CD19 + B cells subsets between patients (SP, n = 48; SD, n = 23; LD, n = 25; FF, n = 33; LF, n = 15.) and HC (n = 25). Error bars represent mean+-SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS P >= 0.05.

Supportive validation

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Experimental details: Figure 3. Ontogenic timing influences gammadeltaT17 generation (A) Flow cytometry analysis of CD27 expression by gammadelta T cells in lymph nodes and lung of fetal-, neonatal-, or adult-induced RBPJ ind mice (left); right panels show percentages. (B) Flow cytometry analysis of IL-17 production by gammadelta T cells in lymph nodes and lung of mice as in (A) stimulated with PMA and ionomycin in vitro (left); right panels show percentages. Data are representative of three independent experiments. Data are presented as means +- standard deviation of three independent experiments. n = 3 mice per group. **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA). (C) Flow cytometry analysis of IL-17 production by gammadelta T cells in lung of control, fetal-, or adult-induced RBPJ ind mice after TDM challenge (top); bottom panels show percentages and numbers. Data are representative of three independent experiments. Data are presented as means +- standard deviation of three independent experiments. n = 6 mice for control; n = 3 mice per fetal- and adult-induced groups. Statistical analyses between groups are found in Figure S2D .

Supportive validation

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Experimental details: Figure 2. ai-mAb-specific B cell sorting and sequencing (A) ai-mAbs fluorescently labeled with either phycoerythrin (PE) or allophycocyanin (APC) were used as bait to single-sort CD19 + CD20 + IgD + IgM + CD27 - B cells from PBMCs obtained from healthy, HIV-1-negative donors. VH and VL transcripts were recovered using RT-PCR, and amplicons were Sanger sequenced. (B and C) Frequencies of B cells expressing VH1-2 HC (B) and 5-aa CDRL3 (C) were compared between the different ai-mAbs. Frequencies in total unselected B cells were assessed as control. Data in (B) and (C) are presented as mean +- SD. All frequency data are given in Table S2 .

Supportive validation

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Experimental details: Figure 6 Primary and secondary status of infection is linked to B cell/antibody readouts (A) NT 50 values of plasma collected at V1-V5 against DENV-1 (left column) or DENV-2 (right column) and for primary patients (top row, blue) or secondary patients (bottom row, red). Each set of connected dots represents 1 patient. (B) Binding of plasma antibodies from V1-V5. ELISA plates were coated with E protein (mostly monomeric, top row) or with UV-inactivated polyethylene glycol (PEG)-precipitated DENV (UV-DENV) (bottom row). E protein ELISA: line indicates the lowest dilution tested (1:500). Endpoint titers for the negative control plasma were 500. UV-DENV ELISA: line indicates the endpoint titer of the negative control plasma (2,500). Lowest dilution tested was 1:100. Primary: blue, secondary: red. Means +- SD are indicated. (C) Frequency of plasmablasts (CD27 + CD38 + ) among CD19 + B cells at V2 for primary and secondary patients. ***p = 0.0007, unpaired t test. Bars indicate the mean. (D) Frequencies of IgA + , IgM + , and IgG + (IgA - IgM - ) plasmablasts during acute disease, stratified according to hospitalization status. Bars indicate the median. (E) IgA expressed at the surface by the plasmablasts in (C); ****p < 0.0001, unpaired t test. Bars indicate the mean (F) Frequency of HLA-DR + cells among CD8 T cells at V2 in primary and secondary patients. **p = 0.002, unpaired t test. Bars indicate the mean. (G) Frequency of CXCR3 + Tfh (circle) or CXCR3 - Tfh (triangle) among CD

Supportive validation

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Experimental details: Figure 1. Arrested NK cell maturation in Id2 Delta/Delta mice. (A) Flow cytometry showing gating strategy for BM NK cells in Ctrl and Id2 Delta/Delta mice. Top panels show lineage (Lin; CD3e)/PI versus NK1.1 on lymphoid cells in the BM. Lower panels show NK1.1 versus CD49b on Lin/PI-NK1.1 + cells. The percentage of cells in the gate is indicated. (B) Summary of the number of NK cells per 10 8 BM cells. Bar represents the average +- SEM. Ctrl (white) and Id2 Delta/Delta (blue). Each circle represents one mouse. (C) Same as A but for spleen. (D) Same as B but for spleen. (E) CD49b + NK1.1 + NK cells from the BM (top) or spleen (bottom) were examined for expression of CD27 and CD11b by flow cytometry. The percentage of cells in each quadrant is indicated. (F and G) Summary of the number of CD27 + CD11b - , CD27 + CD11b + , and CD27 - CD11b + NK cells per 10 8 BM cells or per spleen ( n = 5 for BM and n = 3 for spleen [Sp]; data are from independent experiments). (H and I) RNA expression for the indicated genes in Ctrl or Id2 Delta/Delta determined by RNA-seq and expressed as normalized reads. Data are from three biological replicates. Error bars represent SD. Statistical significance was determined by two-tailed unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Supportive validation

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Experimental details: Figure 2. Dysregulation of Tcf7 /TCF1 in Id2 Delta/Delta NK cells. (A) Tcf7 mRNA in Ctrl and Id2 Delta/Delta NK cells by RNA-seq expressed as normalized reads. Data are from three biological replicates. Error bars represent SD. (B) TCF1 in CD27 + CD11b - NK cells from BM and spleen from Ctrl and Id2 Delta/Delta mice determined by flow cytometry. The MFI of TCF1 is indicated for Ctrl (black) and Id2 Delta/Delta (blue; n = 4 for BM and n = 3 for spleen; data are from independent experiments). (C and D) Summary of relative TCF1 MFI from BM and spleen. MFI of Ctrl NK cells was set as 1 in each experiment. (E) CD49b + -enriched BM NK cells were cultured in IL-15 (20 ng/ml) for 6 d before flow cytometry analysis for TCF1 in NK1.1 + CD49b + cells. The MFI for TCF1 in Ctrl (black) and Id2 Delta/Delta (blue) is shown. (F) Summary of relative TCF1 MFI in IL-15-cultured NK cells. MFI of Ctrl NK cells was set as 1 in each experiment ( n = 4; data are from independent experiments). Error bars represent SEM (C, D, and F). (G) Chromatin accessibility surrounding the Tcf7 gene as determined by ATAC-seq on CD27 + CD11b - NK cells (). Ctrl (black) and ID2-deficient (blue) NK cells. (H) Enhanced view of the indicated region of the Tcf7 intron showing increased chromatin accessibility in ID2-deficient CD27 + CD11b - NK cells. (I) The same region as in H but showing nucleosome depletion. (J) E protein ChIP was performed on chromatin isolated from Ctrl or Id2 Delta/Delta BM NK cells and amplified

Supportive validation

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Experimental details: Figure 3. Deletion of Tcf7 in mature NK cells impacts their numbers and maturation. (A) Flow cytometry for BM NK cells in Ctrl and Tcf7 Delta/Delta mice. Top panels show lineage (Lin)/PI versus NK1.1 on lymphoid cells. Bottom panels show NK1.1 versus CD49b on Lin/PI-NK1.1 + cells. The percentage of cells in the gated region is indicated. (B) Summary of the number of NK cells per 10 8 BM cells in Ctrl (white) and Tcf7 Delta/Delta (maroon). Bars represent the average +- SEM. Each circle represents one mouse ( n = 5). (C) Same as A but for spleen. (D) Same as B but for spleen ( n = 4). (E) NK1.1 + CD49b + NK cells from BM (top) or spleen (Sp; bottom) were examined for expression of CD27 and CD11b by flow cytometry. Numbers indicate the percentage of cells in the respective quadrant. (F and G) Summary of the percentage of CD27 + CD11b - , CD27 + CD11b + , and CD27 - CD11b + NK cells among CD49b + NK1.1 + NK cells in BM and in spleen ( n = 3; all flow cytometry data are from independent experiments). (H) RNA expression for the indicated genes in Ctrl or Tcf7 Delta/Delta NK cells determined by RNA-seq and expressed as normalized reads. Data represent three biological replicates. Error bars represent SD. Statistical significance was determined by two-tailed unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Supportive validation

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Experimental details: Figure 5. TCF1 is required for the arrested maturation of Id2 Delta/Delta NK cells. (A and C) BM and spleen from Id2 Delta/Delta Tcf7 Delta/Delta , Id2 Delta/Delta , and Ctrl mice were analyzed by flow cytometry for NK cells. The top panels show lineage (Lin) + PI versus NK1.1 + on lymphoid cells, and the bottom panels show NK1.1 versus CD49b on Lin/PI - NK1.1 + NK cells. Numbers are the percentage of cells in the indicated gates ( n = 5). (B and D) Summary of data shown in A and C, respectively, for NK cell numbers per 10 8 cells. (E) CD49b + NK1.1 + NK cells from BM (top) and spleen (Sp; bottom) were analyzed by flow cytometry for CD27 and CD11b. Numbers indicate the percentage of cells in the respective quadrant ( n = 6). (F and G) Summary of data shown in E for BM (F) and spleen (G) for frequencies of different NK cell subsets. Error bars represent SEM (B, D, F, and G). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test (B, D, F, and G). (H) Log2FC for Id2 Delta/Delta /Ctrl or Id2 Delta/Delta Tcf7 Delta/Delta /Ctrl determined from RNA-seq. Data are from comparisons of three biological replicates. Statistical significance determined after adjustment for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001.

Supportive validation

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Experimental details: Figure 6. TCF1 deficiency restores NK cell surface receptor expression in Id2 Delta/Delta NK cells. (A) Flow cytometry plots of different receptors on NK cells of BM (top) and spleen (Sp; bottom) from Id2 Delta/Delta Tcf7 Delta/Delta (red), Id2 Delta/Delta (blue), and Ctrl (black) mice. Numbers are the percentage of cells in the indicated gates. Data represent three to six independent experiments ( n = 3-6 for each group). (B) Summary of data shown in A. MFI of Ctrl NK cells was set as 1 in each experiment for CD27 and IL-4Ralpha. Error bars represent SEM. Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. (C and E) BM and spleen tSNE analysis showing expression of CD27 or CD11b on Ctrl NK1.1 + CD49b + NK cells and the overlay of the two markers. Data are representative of two experiments. (D and F) BM and spleen tSNE analysis on NK1.1 + CD49b + NK cells for each of the indicated genotypes. Plots were generated using surface receptors (NK1.1, CD49b, CD27, CD11b, CD49a, KLRG1, CD146, SLAMF6, CD226, IL4Ra, and CXCR3). Data are representative of two experiments.

Supportive validation

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Experimental details: Figure S1. Alterations in gene expression in Id2 Delta/Delta BM NK cells. (A) CD49b + -enriched NK cells from BM were culture in 20 ng/ml IL-15 for 6 d, and then NK1.1 + CD49b + NK cells were analyzed for CD27 and CD11b by flow cytometry. Numbers indicate the percentage of cells in the respective quadrant ( n = 4 from four independent experiments). (B) Summary of relative CD27 MFI. MFI of Ctrl NK cells was set as 1 in each experiment. Error bars represent SEM. Statistical significance was determined by two-tailed unpaired t test. ***, P < 0.005. (C) Volcano plot showing Log2FC versus adj. P value for RNA-seq data from Id2 Delta/Delta and Ctrl NK cells. Data are combined from three biological replicates. (D and E) Example of enrichment data from GSEA of Ctrl and Id2 Delta/Delta RNA-seq data. FDR, false discovery rate; NES, normalized enrichment score.

Supportive validation

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Experimental details: Figure S2. Altered gene expression in Tcf7 Delta/Delta BM NK cells. (A) Sequence of the intronic region of Tcf7 that shows increased accessibility in ID2-deficient BM CD27 + CD11b - NK cells. (B) Schematic representation of the overlap between regions with altered chromatin accessibility in ID2-deficient CD27 + CD11b - NK cells by ATAC-seq and TCF1 binding sites in CD8 T cells determined by ChIP-seq (Gene Expression Omnibus accession no. GSE73239 ). Regions with increased accessibility by ATAC-seq are shown in yellow, decreased accessibility in peach, and no change in orange. TCF1 binding sites are significantly enriched in ATAC-seq regions that gain accessibility in ID2-deficient cells. P < 1.846 10-66 (Fisher's exact test). (C) Volcano plot showing Log2FC versus adj. P values for RNA-seq data from Tcf7 Delta/Delta and Ctrl NK cells. Data were generated from three biological replicates. (D) GSEA for Ctrl versus Tcf7 Delta/Delta NK cell RNA expression. (E) Table showing differentially expressed genes from RNA-seq data shown in C . (F) Flow cytometry plots of different proteins on NK cells of spleen from Tcf7 Delta/Delta and Ctrl mice ( n = 3-4 for each group from independent experiments). (G) Summary of the data in F. Error bars represent SEM. Statistical significance was determined by two-tailed unpaired t test. *, P < 0.05; **, P < 0.01. FDR, false discovery rate; NES, normalized enrichment score.

Supportive validation

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Experimental details: Figure S3. TCF1 deficiency partially rescues ID2-deficient NK cell maturation in the BM and liver. (A) CD49b + -enriched NK cells from BM were culture in 20 ng/ml IL-15 for 6 d, after which NK1.1 + CD49b + cells were analyzed for CD27 and CD11b by flow cytometry. Numbers indicate the percentage of cells in the respective quadrant ( n > 4 from independent experiments). (B) Summary of CD27 MFI for n = 4 in A. MFI of Ctrl NK cells was set as 1 in each experiment. (C) Liver from Id2 Delta/Delta Tcf7 Delta/Delta , Id2 Delta/Delta , and Ctrl mice were analyzed by flow cytometry for NK and ILC1 cells. The top panels were from lymphocyte gate, and the bottom panels were from lineage (Lin)/PI - NK1.1 + gate. NK cells are defined as NK1.1 + CD49b + CD49a - , and ILC1 cells are defined as NK1.1 + CD49b - CD49a + . Numbers are the percentage of cells in the indicated gates ( n = 3 from three independent experiments). (D and E) Summary of data shown in C for NK cell and ILC1 cell numbers per 10 8 cells. (F) NK1.1 + CD49b + CD49a - NK cells from the liver were analyzed by flow cytometry for CD27 and CD11b. Numbers indicate the percentage of cells in the respective quadrant ( n = 3 from three independent experiments). (G) Summary of data shown in F for frequencies of different NK cell subsets. (H) Flow cytometry plots of KLRG1 expression in Id2 Delta/Delta Tcf7 Delta/Delta , Id2 Delta/Delta , and Ctrl mice. Numbers indicate the percentage of cells in the respective quadrant ( n = 3; data ar

Supportive validation

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Experimental details: Figure S4. Heterozygous deletion of Tcf7 restores maturation of Id2 Delta/Delta NK cells to the CD27 + CD11b + stage. (A) Flow cytometry showing CD27 and CD11b on CD3epsilon - NK1.1 + DX5 + NK cells from the BM (top row) or spleen (bottom row) of Ctrl, Id2 Delta/Delta (blue), and Id2 Delta/Delta Tcf7 Delta/+ (green) mice ( n = 4 from four independent experiments). (B) Summary of the frequency of each NK cell subset in CD3epsilon - NK1.1 + DX5 + NK cells from the BM (top) or spleen (bottom) of Ctrl, Id2 Delta/Delta (blue), and Id2 Delta/Delta Tcf7 Delta/+ (green) mice. Error bars are SEM. *, P < 0.05 determined by one-way ANOVA with Tukey's multiple comparisons test. (C) Intracellular expression of TCF1 or TBET in BM or spleen NK cells from each of the indicated mouse strains. Numbers are MFI. One representative experiment is shown ( n = 2 for Id2 Delta/Delta Tcf7 Delta/+ NK cells and n > 4 for Ctrl, Id2 Delta/Delta , and Id2 Delta/Delta Tcf7 Delta/Delta NK cells).

Supportive validation

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Experimental details: Figure 3. Pik3cd E1020K/+ mice fail to develop a robust T CM population (A-F) Viable CD8 + splenocytes from mice infected with LCMV Armstrong (n = 2, 4-5/group/time point). (A-C) NP396-specific CD8 + cells day 15 p.i. (A) CD127 and KLRG1 expression (left: representative staining; right: %KLRG1 + CD127 - ). (B) Representative histogram of CD27 (top) and CD127 (bottom). (C) GzmB and TCF1 staining. Middle: TCF1 MFI; right: GzmB MFI. (D-F) NP396-specific CD8 + cells day 58 p.i. (D) CD44 and CD62L staining. Representative flow (left), % CD44 hi CD62L lo cells (right). (E) CD127 histograms: CD44 hi CD62L lo (top) and CD44 hi CD62L + (bottom). (F) TCF1 MFI. (G) TCF1 staining of allo-reactive CD8 + cells from healthy controls and patients with APDS (n = 2, representative histogram). (H-L) Mice were infected with X31 and challenged with PR8 (n = 2, 3-5 mice/genotype/time point). (H) Infection outline. (I) PA224-specific CD8 + cell numbers. (J) IFN-gamma and TNF-alpha from day 8 cells stimulated with either PA 224-233 (left) or anti-CD3 plus anti-CD28 (right). (K) NP366-specific CD8 + T cell numbers. (L) IFN-gamma and TNF-alpha from day 35 cells stimulated with either NP 366-374 (left) or anti-CD3 plus anti-CD28 (right). (M-O) OT-1 cells were transferred into congenic hosts, infected with influenza X31-OVA and challenged with PR8-OVA (n = 2, 3 mice/genotype/time point). (M) Outline. (N) Viable OT-1 cell numbers. (O) TCF1 and GzmB expression in OT-1 cells on day 35. Representative exper

Supportive validation

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Experimental details: Figure 1 Dynamic Tsc1 expression following IL-15 stimulation. ( a - c ) Quantitative reverse transcription-PCR (RT-PCR) analysis of the indicated genes in sorted CD3 - NK1.1 + cells from the spleen of WT mice before and after stimulation with 25 ng ml -1 IL-15 for 18 h ( a ), various concentration of IL-15 ( b ), or at the indicated time points ( c ). ( d ) Intracellular phosphorylated S6 in sorted NK cells after stimulation with 25 ng ml -1 IL-15 was detected by flow cytometry at the indicated time points, and the mean fluorescence intensity was calculated. ( e ) Tsc1 messenger RNA (mRNA) expression was analysed by quantitative RT-PCR in sorted CD3 - NK1.1 + cells after stimulation with 25 ng ml -1 IL-15 for 18 h in the presence of DMSO or rapamycin (10 nM). ( f ) Analysis of Tsc1 mRNA expression in sorted T, B and NK cells by quantitative PCR. (g) Analysis of Tsc1 mRNA expression in CLP, NKp and immature NK cells (iNK), and NK cell subsets, including CD27 - CD11b - (DN), CD27 + CD11b - (CD27 SP), CD27 + CD11b + (DP) and CD27 - CD11b + (CD11b SP), by quantitative PCR. The results were normalized to beta- actin ( f , g ) or are presented relative to expression in untreated cells, which was set as 1 ( a - c , e ). Value represent mean+-s.d. * P