t reg cell differentiation kit  (R&D Systems)

Journal: Scientific Reports

Article Title: Inflammatory Responses Reprogram T REGS Through Impairment of Neuropilin-1

doi: 10.1038/s41598-019-46934-x

Figure Lengend Snippet: PMMA particles induce NF-κB activity and alter bone marrow cellularity toward reduced immunosuppression. ( a ) PMMA particles induced local NF-κB activation in immune cells. Total cells were isolated from the bone marrow of RelA-Luciferase reporter mice post PMMA intra-tibial injection. After removal of red blood cells, mononucleated cells from bone marrow of separated femurs and tibias were lysed to assess luciferase activity, which was normalized by protein concentration determined by standard BCA assay. ( b ) Increase of myeloid progenitor cells in the bone marrow of mice 2 days post intra-tibial injection of PMMA particles revealed by flow cytometric analysis. 1 × 10 7 mononucleated bone marrow cells were stained with FACS antibodies to assess myeloid progenitor populations including LSK HSCs, CMPs and GMPs. ( c ) Decreased frequency of bone marrow CD4 + CD25 + Foxp3 + T REG by intra-tibial PMMA injection. ( d ) Alterations of bone marrow myeloid lineages. Monocytic myeloid cells are marked as CD11b + Gr-1 − and granulocytic cells as CD11b + Gr-1 + . ( e , f ) Assessment of osteoclastogenic potential of whole bone marrow cells (WBM) by ex vivo osteoclastogenesis assay (OCgenesis) 2 days after intra-tibial injection of PMMA. 50,000, 100,000 or 200,000 total bone marrow cells were cultured in 96-well plates supplemented with CMG and RANKL to achieve optimal cell density for osteoclast formation. After 4 days of culture, cells were fixed before subjected TRAP staining to visualize osteoclasts. Cells with expanded cytoplasm and more than 3 nuclei were counted as matured osteoclasts. ( e ) Representative images and quantification of the number of multi-nucleated (MNC) osteoclasts per well counted from triplicates of three independent experiments. All columns in the graphs were represented as mean ± SD. *p < 0.05; **p < 0.005 or as indicated by Student T-test.

Article Snippet: To transduce T REGS , 1 millions of freshly MACS-isolated naïve CD4+ T cells from mouse spleen were incubated overnight with 3 ml of retroviral stock supplemented with reagents from the T REG Cell Differentiation Kit (R&D Systems) and 10ug/ml of polybrene.

Techniques: Activity Assay, Activation Assay, Isolation, Luciferase, Injection, Protein Concentration, BIA-KA, Staining, Ex Vivo, Cell Culture

Journal: Scientific Reports

Article Title: Inflammatory Responses Reprogram T REGS Through Impairment of Neuropilin-1

doi: 10.1038/s41598-019-46934-x

Figure Lengend Snippet: PMMA particles induce NF-κB activity and modulate extra-medullary hematopoiesis in the spleen. ( a ) PMMA particles induced NF-κB activation in immune cells. Total cells were isolated from the spleen from post PMMA intratibially injected animals. After removal of red blood cells, mononucleated cells were lysed to assess luciferase activity, which was normalized by protein concentration determined by standard BCA assay. ( b ) CD4 + T helper were further fractionated before measurements of luciferase activity normalized either by total protein input or cell number (not shown). ( c ) Decreased frequency of bone marrow CD4 + CD25 + Foxp3 + T REG in the spleen of PMMA injected animals. ( d ) Alterations of myeloid lineages. All columns in the graphs were represented as mean ± SD. *p < 0.05 by Student T-test.

Article Snippet: To transduce T REGS , 1 millions of freshly MACS-isolated naïve CD4+ T cells from mouse spleen were incubated overnight with 3 ml of retroviral stock supplemented with reagents from the T REG Cell Differentiation Kit (R&D Systems) and 10ug/ml of polybrene.

Techniques: Activity Assay, Activation Assay, Isolation, Injection, Luciferase, Protein Concentration, BIA-KA

Journal: Scientific Reports

Article Title: Inflammatory Responses Reprogram T REGS Through Impairment of Neuropilin-1

doi: 10.1038/s41598-019-46934-x

Figure Lengend Snippet: Increase of myeloid progenitors in response to PMMA is transient, while reduction of regulatory T cells is prolonged. ( a ) Frequency of bone marrow myeloid progenitor LSK, ( b ) CMP and ( c ) GMP 2, 4 and 7 days after intra-tibial injection of PMMA. ( d ) Frequency of T REG in the bone marrow (BM) and ( e ) in the spleen (Spl) over time. ( f ) Frequency of granulocytes in the mononuclear peripheral blood cells (PB/MNCs) over time. ( g ) IHC for Luciferase and FoxP3 from Control and PMMA-treated conditions. Arrows point to reactive staining ( h ) Quantification of normalized NF-κB reporter activity in WBM cells isolated from femur and tibia 7 days after intra-tibial (i.t.) injection of PMMA. # denotes femur adjacent to injected tibia. ( i ) Assessment of osteoclastogenic potential of WBM cells by ex vivo osteoclastogenesis assay (OCgenesis) 7 days after intra-tibial injection of PMMA. ( j ) % of CD4 + CD25 + FoxP3 lo RORγt + T cells in the bone marrow (BM) of mice treated as described in H-I. All columns in the graphs were represented as mean ± SD. *p < 0.05; **p < 0.005 by Student T-test.

Article Snippet: To transduce T REGS , 1 millions of freshly MACS-isolated naïve CD4+ T cells from mouse spleen were incubated overnight with 3 ml of retroviral stock supplemented with reagents from the T REG Cell Differentiation Kit (R&D Systems) and 10ug/ml of polybrene.

Techniques: Injection, Luciferase, Control, Staining, Activity Assay, Isolation, Ex Vivo

Journal: Scientific Reports

Article Title: Inflammatory Responses Reprogram T REGS Through Impairment of Neuropilin-1

doi: 10.1038/s41598-019-46934-x

Figure Lengend Snippet: Increase of T H 17 immunity, upregulation of inflammatory factors produced by effector T cells and recapitulation of the in vivo PMMA effect on T REG by ex vivo cultures. ( a – c ) CD4 + CD25 − effector T cells (T EFF ) were enriched by magnetic bead sorting (MACS) and lysed in Trizol reagent for RNA isolation. After cDNA synthesis through reverse transcription (RT), quantitative polymerase chain reaction (qPCR) was performed to assess mRNA expression of T H 17 markers including IL-17A, RORγt and RUNX1. ( d – f ) Expression of proinflammatory/pro-osteoclastogenic cytokines including M-CSF, RANKL and TNFα were also measured by qRT-PCR. Expression of tubulin was used as loading control. ΔCq values were calculated by Cq value of each given gene divided by that of tubulin. Plots were generated by values that were normalized with mock PBS injected control group. ( g – i ) Frequencies of CD4 + CD25 + Foxp3 + T REG in whole bone marrow (WBM), whole spleen (WSpl) and while lymph node (WLN) cells were assessed by flow cytometry after overnight incubation with 0.4% PMMA. ( j – l ) Frequencies CD4 + CD25 + CD44 + IL-17A + T EFF were also analyzed. All columns in the graphs were represented as mean ± SD. *p < 0.05; **p < 0.005 by Student T-test.

Article Snippet: To transduce T REGS , 1 millions of freshly MACS-isolated naïve CD4+ T cells from mouse spleen were incubated overnight with 3 ml of retroviral stock supplemented with reagents from the T REG Cell Differentiation Kit (R&D Systems) and 10ug/ml of polybrene.

Techniques: Produced, In Vivo, Ex Vivo, Isolation, cDNA Synthesis, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Control, Generated, Injection, Flow Cytometry, Incubation

Journal: Scientific Reports

Article Title: Inflammatory Responses Reprogram T REGS Through Impairment of Neuropilin-1

doi: 10.1038/s41598-019-46934-x

Figure Lengend Snippet: Inhibition of osteoclastogenesis by T REG in the presence of PMMA is Nrp1-mediated event. ( a ) MACS-purified T REG were either co-cultured with bone marrow derived macrophages (BMMs) or placed in the upper chambers of transwell cultures with BMMs in the lower chambers. RANKL and M-CSF were added at the beginning of cell culture and again after 2 days to promote osteoclastogenesis. PMMA particles were added to BMMs or T REGS as indicated. After 4 days, T REG cells were separated from cultures before TRAP staining was performed to visualize osteoclast formation. Results from BMM-T REG co-cultures and BMM-T REG transwell cultures are marked accordingly. ( b – e ) Isolated T REG from co-cultures and transwell upper chambers were lysed in Trizol reagent for RNA isolation. cDNAs were subsequently generated for qPCR analysis. ( f ) Immunostaining for Nrp1 in bone sections from control and PMMA-treated mice. ( g ) Osteoclast differentiation (+/−PMMA treated T REGS ) as described in panel A in the absence or presence of intact T REGS or T REGS transduced with scrambled (scr) shRNA or Nrp-1 shRNA. ( h ) Osteoclast differentiation (as described above) in the absence or presence of vehicle or PMMA-treated T REGS . Some conditions include overexpression of retroviral Nrp-1 in T REGS (as indicated). ( i ) Representative images of osteoclast cultures quantified in panel h. All columns in the graphs were represented as mean ± SD. *p < 0.05; **p < 0.005; ***p < 0.001 by Student T-test.

Article Snippet: To transduce T REGS , 1 millions of freshly MACS-isolated naïve CD4+ T cells from mouse spleen were incubated overnight with 3 ml of retroviral stock supplemented with reagents from the T REG Cell Differentiation Kit (R&D Systems) and 10ug/ml of polybrene.

Techniques: Inhibition, Purification, Cell Culture, Derivative Assay, Staining, Isolation, Generated, Immunostaining, Control, Transduction, shRNA, Over Expression, Retroviral