Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: Analysis of the WE mutant mouse phenotype and identification of the causative mutation. (A) Selection of the ENU-148 pedigree using a functional screen based on an in vivo NK cell–killing assay. Splenocytes from WT and b2m -deficient ( b2m −/− ) mice were isolated, stained with two different concentrations of the fluorescent dye CFSE, and injected i.v. into the indicated recipient mice (C57BL/6 control mice or G3 animals from pedigree ENU-148). 2 d after transfer, the relative frequency of the CFSE high and CFSE low populations present in the blood of recipient mice was assessed by flow cytometry, and the percentages of b2m −/− cell killing were calculated. Three G3 mice with a defect in NK cell–mediated target cell killing were selected (red circle) and crossed to WT animals to generate a colony of mutant mice. An autosomal-recessive transmission of the mutation was observed, and the phenotype of affected animals was called WE. (B) In the ENU-148 colony, some mice showed progressive hair hypopigmentation. The pictures show a typical example of this phenotype for mice aged between 6 wk and >10 mo, as indicated. (C) NK cell counts in the blood of control (C57BL/6J) mice and mice from the pedigree ENU-148 presenting or not with hair hypopigmentation (gray and black, respectively). Each dot represents the results obtained for one mouse ( n = 31–82, Kruskal–Wallis statistical test). (D) ENU-148 mice were segregated depending on their Bcl2 genotype, and the NK cell counts in peripheral blood were measured. Each dot represents the results obtained from an individual mouse ( n = 18–79, Kruskal–Wallis statistical test). (E) Enumeration of CD4 + , CD8 + T cells and B cells in the blood of Bcl2 +/+ and Bcl2 WE/WE mice ( n = 18–67, Mann–Whitney statistical test). (F) Comparison of the size of the spleens from 9-wk-old Bcl2 +/+ , Bcl2 WE/+ , and Bcl2 WE/WE mice. (G) Enumeration of monocytes, granulocytes, and eosinophils in the blood of Bcl2 +/+ and Bcl2 WE/WE mice ( n = 18–67, Mann–Whitney statistical test). (H) Percentage of NK cells in the spleens of Bcl2 +/+ and Bcl2 WE/WE mice ( n = 28, Mann–Whitney statistical test). Each dot corresponds to the data obtained from an individual mouse. Horizontal lines indicate the mean. *, P < 0.05; **, P < 0.01; and ****, P < 0.0001.

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Mutagenesis, Selection, Functional Assay, In Vivo, Isolation, Staining, Injection, Control, Flow Cytometry, Transmission Assay, MANN-WHITNEY, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: Identification of a point mutation in the Bcl2 gene on Chr1 as the causative mutation for the WE phenotype

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: Bcl2 expression in WE splenocytes. (A) Schematic representation of the BCL2 protein. The point mutation responsible for the WE phenotype induces a change of tyrosine to cysteine at amino acid position 18 in the BH4 domain of the BCL2 protein. (B) Bcl2 mRNA expression was assessed by quantitative RT-PCR. The fold change in the expression of Bcl2 transcripts in Bcl2 WE/WE versus Bcl2 +/+ splenocytes is shown ( n = 5). Each dot on the figure corresponds to the data obtained for a single experiment. (C) Analysis of BCL2 protein expression by immunoblotting in splenocytes from Bcl2 WE/WE , Bcl2 WE/+ , and Bcl2 +/+ mice (representative of three independent experiments). Probing for β-ACTIN was used as a loading control. (D) BCL2 expression was analyzed by flow cytometry. Representative intracellular staining of BCL2 in splenic NK cells from Bcl2 WE/WE and Bcl2 +/+ mice are shown (data shown are representative of three independent experiments).

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Western Blot, Control, Flow Cytometry, Staining

Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: NK cells in the BM and the spleen of WE mice display an immature phenotype. (A, left) Representative flow cytometric profiles of BM-derived NK cells (CD122 + CD3 − ) from Bcl2 +/+ and Bcl2 WE/WE mice. (right) NK cell counts in the BM of Bcl2 +/+ and Bcl2 WE/WE mice ( n = 23, Mann–Whitney statistical test). (B, left) Representative flow cytometric profiles of BM NK cell maturation states from Bcl2 +/+ and Bcl2 WE/WE mice: NK1.1 − NKp46 − , NK1.1 + NKp46 − , NK1.1 + NKp46 + CD27 − CD11b − , NK1.1 + NKp46 + CD27 + CD11b − (immature), NK1.1 + NKp46 + CD27 + CD11b + (mature 1), and NK1.1 + NKp46 + CD27 − CD11b + (mature 2). (right) The relative cell counts of subsets of CD122 + CD3 − BM NK cells is shown based on their maturation states ( n = 23, paired Student’s t test). (C, left) Representative flow cytometric profiles of splenic NK cells (CD3 − NKp46 + ) from Bcl2 +/+ and Bcl2 WE/WE mice. (right) NK cell counts in spleens from Bcl2 +/+ and Bcl2 WE/WE mice ( n = 25, Mann–Whitney statistical test). (D) Representative flow cytometric profiles (left) and relative frequency of NK cell subsets (right) of splenic NK cells from Bcl2 +/+ and Bcl2 WE/WE mice: NK1.1 + NKp46 + CD27 + CD11b − (immature), NK1.1 + NKp46 + CD27 + CD11b + (mature 1), NK1.1 + NKp46 + CD27 − CD11b + (mature 2; n = 20, two-way ANOVA with Bonferroni correction statistical test), and CD43 + expression ( n = 20, Mann–Whitney statistical test). Each dot represents the data obtained from an individual mouse. Horizontal lines indicate the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Derivative Assay, MANN-WHITNEY, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: A cell-intrinsic role of BCL2 in NK cell homeostasis. (A) Mixed BM chimera experiments were performed. WT CD45.1 + BM cells were mixed at a 1:1 ratio with either CD45.2 + Bcl2 WE/WE or CD45.2 + Bcl2 +/+ BM cells and transferred into lethally irradiated WT CD45.1 recipient. Chimeric mice were analyzed 12 wk later. (left) Representative flow cytometric profiles of NK cell reconstitution in the spleens of chimeric WT/Bcl2 +/+ (top) and WT/Bcl2 WE/WE (bottom) mice stained with anti-CD45.1 and anti-CD45.2 mAbs. (right) The percentages of CD45.1 + and CD45.2 + NK cells in the two groups of chimeras are shown. (B, left) Representative flow cytometric profiles of spleen NK cells (TCRβ − NKp46 + ) from Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice. (right) NK cell counts in the spleens of Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice ( n = 10, Mann–Whitney statistical test). (C) Representative flow cytometric analysis (left) and relative counts of NK cell subsets in the spleens of Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice were analyzed (middle): NK1.1 + NKp46 + CD27 + CD11b − (immature), NK1.1 + NKp46 + CD27 + CD11b + (mature 1), NK1.1 + NKp46 + CD27 − CD11b + (mature 2; n = 4, two-way ANOVA with Bonferroni correction statistical test). CD43 expression on NK cells is also shown (right; n = 6, Mann-Whitney statistical test) of Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice. (D) T and B cell counts in the spleens of Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice ( n = 6, two-way ANOVA with Bonferroni correction statistical test). Each dot corresponds to the data obtained from an individual mouse. Horizontal lines indicate the mean. **, P < 0.01; and ****, P < 0.0001.

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Irradiation, Staining, MANN-WHITNEY, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: NK cell responsiveness in Bcl2 WE/WE mutant mice. (A, left) Representative flow cytometric profiles of IFN-γ and CD107 expression in Bcl2 +/+ or Bcl2 WE/WE NK cells after 4 h of NK1.1 stimulation. (middle and right) Percentage of IFN-γ + or CD107 + NK cells after 4 h of activation with an Ig isotype-matched control mAb (IC), anti-NK1.1 mAb, IL-12, and IL-18 or PMA plus ionomycin (PMA-iono; n = 8–11, two-way ANOVA with Bonferroni correction statistical test). Each dot corresponds to the data obtained from a single mouse. (B) Representative flow cytometric profiles of IFN-γ and CD107 expression in Bcl2 +/+ or Bcl2 WE/WE LAK cells, after 4 h of incubation with YAC-1 cells. Representative of four mice in two experiments. (C) Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice were injected i.v. with E0771-LMB mCherry + breast cancer cells. Lung metastases were measured 14 d later by IVIS Spectrum in vivo imaging system (PerkinElmer) for mCherry fluorescence (vertical axis: total radiant efficiency [(p/s)/ÌW/cm 2 ] × 1e 6 ). P = 0.008, n = 5, Mann–Whitney statistical test. Horizontal lines indicate the mean. *, P < 0.05.

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Mutagenesis, Expressing, Activation Assay, Control, Incubation, Injection, In Vivo Imaging, Fluorescence, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: Impaired NK cell survival in BCL2 -deficient mice is restored in inflammatory conditions. (A, left) Representative flow cytometric profiles of Bcl2 +/+ and Bcl2 WE/WE splenic NK cells, stained with dead cell marker (DCM) after 4 h of in vitro culture in complete medium. (right) Percentages of DCM + NK cells ( n = 21, Mann–Whitney statistical test). (B, left) Representative flow cytometric profiles of spleen NK cells (CD3 − NKp46 + ) from Bcl2 +/+ and Bcl2 WE/WE mice 0 or 7 d after MCMV infection. (middle) Increase in NK cell number in the spleen 7 d after MCMV infection ( n = 8, Mann–Whitney statistical test). (right) Spleen NK cell counts 0 or 7 d after MCMV infection in Bcl2 +/+ and Bcl2 WE/WE mice ( n = 8, two-way ANOVA with Bonferroni correction statistical test). (C, left) Representative flow cytometric profiles of spleen NK cells (TCRβ − NKp46 + ) from Ncr1 iCre/+ Bcl2 +/+ , Ncr1 iCre/+ Bcl2 fl/fl , and Ncr1 iCre/+ Mcl1 fl/fl mice injected with PBS or IL-2/S4B6. (right) Spleen NK cell fold increase and counts after PBS or IL-2/S4B6 injection. (D) Percentages of Ki67 + NK cells in Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice after PBS or IL-2/S4B6 injection. Each dot corresponds to the data obtained for an individual mouse. Horizontal lines indicate the mean. *, P < 0.05; **, P < 0.01; and ****, P < 0.0001.

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Staining, Marker, In Vitro, MANN-WHITNEY, Infection, Injection

Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: BCL2 loss is intrinsically linked to an increase of proliferating NK cells. (A, left) Representative flow cytometric profiles of Bcl2 +/+ and Bcl2 WE/WE or Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl splenic NK cells, stained with anti-Ki67 mAb. (right) Percentages of Ki67 + NK cells ( Bcl2 +/+ / Bcl2 WE/WE n = 21, Ncr1 iCre/+ Bcl2 +/+ / Ncr1 iCre/+ Bcl2 fl/fl n = 7, Mann–Whitney statistical test). Each dot represents the data obtained for a single mouse. (B, left) Representative flow cytometric profiles of Ki67 staining in the indicated NK cell subsets: NK1.1 + NKp46 + CD27 + CD11b − (immature), NK1.1 + NKp46 + CD27 + CD11b + (mature 1), and NK1.1 + NKp46 + CD27 − CD11b + (mature 2). (right) The percentages of Ki67 + cells in the indicated NK cell subsets from Bcl2 +/+ and Bcl2 WE/WE mice ( n = 21, two-way ANOVA with Bonferroni correction statistical test). Each dot represents the data obtained for a single mouse. (C) Representative flow cytometric profiles of the liver, BM, and spleen NK cells from Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice stained with anti-BCL2 and anti-Ki67 mAbs. Data are representative of three independent experiments. Horizontal lines indicate the mean. **, P < 0.01; and ****, P < 0.0001.

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Staining, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: NK cells in cell cycle are less dependent on BCL2 for their survival. (A) NK cells from C57BL/6 mice were cultured for 20 h in DMSO or 30 nM, 100 nM, or 300 nM of the BCL2 inhibitor ABT-199. Representative flow cytometric profiles of Ki67 staining on NK cells (left) and percentages of Ki67 + NK cells (middle) are shown ( n = 3–6 Kruskal–Wallis statistical test). Ki67 + and Ki67 − NK cell counts are shown for DMSO or 300 nM ABT-199 culture (right). (B) Ki67 + and Ki67 − NK cell counts are shown for Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice (left, n = 4) and for Bcl2 +/+ and Bcl2 WE/WE mice (right, n = 19 Kruskal–Wallis statistical test). Horizontal lines indicate the mean. ***, P < 0.001; and ****, P < 0.0001. (C and D) Cell numbers and mean division number versus time graphs of C57BL/6 NK cells cultured in the presence of 10 ng/ml IL-15 (C) or 200 ng/ml IL-15 (D) and 0 nM, 30 nM, or 100 nM of the BCL2 inhibitor ABT-199. Error bars indicate SEM. (E) CTV profiles at 69.5 h, with the undivided peak depicted ( n = 1, in triplicate, from pooled spleens of 12 mice).

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Cell Culture, Staining

Journal: The Journal of Experimental Medicine

Article Title: Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

doi: 10.1084/jem.20160869

Figure Lengend Snippet: MCL1 expression is increased in BCL2 -deficient NK cells. (A, left) Representative flow cytometric profiles of MCL1 expression in NK cells from Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl are shown. (right) Expression levels of MCL1 (mean fluorescence intensity [MFI]) in NK cells from Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl ( n = 6), Bcl2 +/+ , and Bcl2 WE/WE mice ( n = 9, Mann–Whitney statistical test). (B, left) Representative flow cytometric analysis of MCL1 expression in Ki67 − and Ki67 + NK cells from Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice. (right) MCL1 expression (MFI) in cycling (Ki67 + ) or quiescent (Ki67 − ) NK cell subsets from Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl ( n = 4) as well as Bcl2 +/+ and Bcl2 WE/WE mice ( n = 8, two-way ANOVA with Bonferroni correction statistical test). (C) MFI of MCL1 expression in NK cells from Ncr1 iCre/+ Bcl2 +/+ and Ncr1 iCre/+ Bcl2 fl/fl mice after PBS or IL-2/S4B6 injection. Each dot corresponds to the data obtained for an individual mouse. (D) Enumeration of NK cells from the spleens of Ncr1 iCre/+ Bcl2 +/+ , Ncr1 iCre/+ Bcl2 fl/fl , Ncr1 iCre/+ Bcl2l11 fl/fl , Ncr1 iCre/+ Mcl1 fl/fl , Ncr1 iCre/+ Bcl2 fl/fl Bcl2l11 fl/fl , and Ncr1 iCre/+ Mcl1 fl/fl Bcl2l11 fl/fl mice. Each dot corresponds to the data obtained for an individual mouse. **, P = 0.008; n = 5 (Mann–Whitney statistical test). (E) Ki67 and MCL1 expression in splenic NK cells from the indicated mice. Histograms are representative of five mice per genotype. Horizontal lines indicate the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

Article Snippet: Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex).

Techniques: Expressing, Fluorescence, MANN-WHITNEY, Injection

Journal: The Journal of Experimental Medicine

Article Title: Hierarchy of Notch–Delta interactions promoting T cell lineage commitment and maturation

doi: 10.1084/jem.20061442

Figure Lengend Snippet: N2 signaling is sufficient to induce T lineage commitment in vitro but not in vivo. (A) Mixed BM chimeric mice were analyzed 8 wk after reconstitution with a 1:2 mixture of WT (CD45.1 + ) and Ctrl ( N1 lox/lox ), N1 −/− , N2 −/− , or N1N2 −/− (CD45.2 + ) BM-derived populations. Representative FACS analysis of thymocytes stained with anti-CD117, -CD44, and -CD25 antibodies after gating on donor (CD45.2 + )-derived lineage-negative cells (top). Representative FACS analysis of thymocytes stained with anti-B220 and -CD19 antibodies after gating on donor (CD45.2 + )-derived lineage-negative cells (bottom). Representative FACS profiles are derived from experiments in which five mice of each genotype were analyzed. (B) BM KLS cells were sorted from Ctrl , N1 −/− , N2 −/− , and N1N2 −/− mice and cultured on OP9-DL1 cells for 10 d (top) and 18 d (bottom). Cells from these cultures were analyzed for the expression of CD44 and CD25 (top) and for the presence of B220 + CD19 + B cells (bottom). Representative FACS profiles are derived from four individual experiments. (C) Deletion PCR analysis for the N1 gene was performed on genomic DNA from sorted CD25 − (corresponding to DN1) and CD25 + (corresponding to DN2/DN3) cells derived from N1 −/− and Ctrl animals cultured for 10 d on OP9-DL1. (D) Semiquantitative RT-PCR for the expression of N1 , N2 , and tubulin was performed on sorted BM KLS cells. Three serial dilutions (threefold) of template RNA are shown for the indicated genes.

Article Snippet: The following monoclonal antibody conjugates were purchased from eBioscience: CD117 (2B8)-PE and -PE-Cy5.5; Sca-1 (D7)-PE and -APC; CD19 (MB-19.1)-PE and (6D5)-PE-Cy5.5; B220 (RA3.6B2)-PE-Cy5.5 and –Alexa Fluor 647; CD44 (IM781)-PE-Cy5.5; CD25 (PC61)-APC; CD4 (L3T4)-PE-Cy5.5; CD45.2 (104)-PE-Cy5.5; TCRβ (H57)-PE and -APC; CD161 (PK136)-FITC; CD90.1 (HIS15)-PE; and CD90.2 (30H12)-PE.

Techniques: In Vitro, In Vivo, Derivative Assay, Staining, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Hierarchy of Notch–Delta interactions promoting T cell lineage commitment and maturation

doi: 10.1084/jem.20061442

Figure Lengend Snippet: N2 is sufficient to specify T lineage progenitors in the spleen after BM transplantation. CD45.2 + Ctrl , N1 −/− , N2 −/− , or N1N2 −/− BM cells were injected into lethally irradiated CD45.1 + hosts. The spleens of host mice were analyzed 12 d after BM transplantation. Representative flow cytometric analyses of donor-derived lineage-negative cells for the expression of CD44 and Thy1.2, and Thy1.2 and CD25, respectively, are shown. Data are representative of four independent experiments.

Article Snippet: The following monoclonal antibody conjugates were purchased from eBioscience: CD117 (2B8)-PE and -PE-Cy5.5; Sca-1 (D7)-PE and -APC; CD19 (MB-19.1)-PE and (6D5)-PE-Cy5.5; B220 (RA3.6B2)-PE-Cy5.5 and –Alexa Fluor 647; CD44 (IM781)-PE-Cy5.5; CD25 (PC61)-APC; CD4 (L3T4)-PE-Cy5.5; CD45.2 (104)-PE-Cy5.5; TCRβ (H57)-PE and -APC; CD161 (PK136)-FITC; CD90.1 (HIS15)-PE; and CD90.2 (30H12)-PE.

Techniques: Transplantation Assay, Injection, Irradiation, Derivative Assay, Expressing