Journal: Glia

Article Title: Inflammatory Mediators Both Directly and Indirectly Promote Microglial Proliferation

doi: 10.1002/glia.70150

Figure Lengend Snippet: CSF2, IL3, and TNFɑ, but not other screened factors, boost microglial proliferation. (A) Schematic representation for microglial immunopanning, culture, and treatment. (B) Bar plot depicting the fold change in percentage of proliferative microglia in response to each screened factor at 10 ng/mL relative to the MGM control. (C) Representative images of CSF2, IL3, or TNFɑ induced microglial proliferation. (D) Bar plot depicting the percentage of proliferative microglia in response to 10 ng/mL CSF2. (E) Bar plot depicting the percentage of proliferative microglia in response to 10 ng/mL IL3. (F) Bar plot depicting the percentage of proliferative microglia in response to 10 ng/mL TNFɑ. (B): N = 3 independent microglial cultures with treatment in triplicate; (D–F): N = 9–12 wells across 3–4 independent microglial cultures. Bars represent mean ± SEM. B: One‐way ANOVA, Dunnett's post hoc comparing fold changes to mean MGM control; (D–F): Unpaired t ‐test; Colors represent independent microglial cultures: Large data points = mean percentage of proliferative microglia per culture, small data points = percentage of proliferative microglia per well. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar = 50 μm. BDNF, Brain‐derived neurotrophic factor; CSF, Colony stimulating factor; CX3CL1, Fractalkine; IFN, Interferon; IGF, Insulin‐like growth factor; IL, Interleukin; LPA, Lysophosphatidic acid; MGM, Microglia growth media; NGF, Nerve growth factor β; NRG1, Neuregulin 1; NT3, Neurotrophin 3; NTS, Neurotensin; S1P, Sphingosine‐1 phosphate; SCF, Stem cell factor; TNFɑ, Tumor necrosis factor ɑ; VEGF, Vascular endothelial growth factor; WNT3a, Wingless‐related int‐3a.

Article Snippet: To determine the concentration of CSF2 in our concentrated ACM, we performed a CSF2 ELISA (R&D Systems MGM00) as per the manufacturer's instructions.

Techniques: Control, Derivative Assay

Journal: Glia

Article Title: Inflammatory Mediators Both Directly and Indirectly Promote Microglial Proliferation

doi: 10.1002/glia.70150

Figure Lengend Snippet: Astrocyte‐secreted CSF2 promotes microglial proliferation in response to IL1ɑ and IL1β stimulation. (A) Plot depicting the percentage of proliferative microglia following 10 ng/mL treatment with each elevated cytokine identified by cytokine assay in microglia media lacking CSF1 and TGFβ2. (B) Plot depicting the concentration of CSF2 protein as determined by ELISA in 50× concentration ACM, IL1ɑ and IL1β ACM. (C) Representative images of 50× concentrated IL1ɑ ACM and IL1β ACM induced microglial proliferation (red = CD11b; white = KI67) and CSF2 neutralization. (D) Plot depicting the percentage of proliferative microglia in response to 50× concentrated UCM, ACM, IL1ɑ ACM with either IgG2A isotype control or ɑCSF2 treatment. (E) Plot depicting the percentage of proliferative microglia in response to 50× concentrated UCM, ACM, IL1β ACM with either IgG2A isotype control or ɑCSF2 treatment. Bars represent mean ± SEM. A: N = 12 wells from 3 independent cultures; (B): N = 4 batches of ACM; (D‐E): N = 9 wells from 3 independent cultures, the same AUCM and ACM controls were used for each of these experiments as they were prepared and tested alongside each respective IL1 ACM. (A, C, D): Colors represent independent microglial cultures: Large data points = mean percentage of proliferative microglia per culture, small data points = percentage of proliferative microglia per well. (A, B, D, E): One‐way ANOVA, Tukey's post hoc to compare differences between all conditions, * p < 0.05. Scale bars = 50 μm. ACM, Astrocyte conditioned media; CSF, Colony stimulating factor; CXCL, C‐X‐C motif chemokine ligand; CX3CL1, Fractalkine; IL, Interleukin; UCM, Unconditioned media.

Article Snippet: To determine the concentration of CSF2 in our concentrated ACM, we performed a CSF2 ELISA (R&D Systems MGM00) as per the manufacturer's instructions.

Techniques: Cytokine Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Control

Journal: Mediators of Inflammation

Article Title: IL-1 β and TNF α Promote Monocyte Viability through the Induction of GM-CSF Expression by Rheumatoid Arthritis Synovial Fibroblasts

doi: 10.1155/2014/241840

Figure Lengend Snippet: Effect of IL-1 β and TNF α on M-CSF, IL-34, and GM-CSF expression in RA SF . SF were stimulated by IL-1 β (25 ng/mL) or TNF α (25 ng/mL) for 24 hours. (a)–(c) RNA was harvested and a qPCR for M-CSF, IL-34, and GM-CSF expression was performed. Results are given as the relative expression using hHPRT gene as housekeeping gene. The bars represent the mean and each plots one biological replicate ( n = 6 patients). (d) GM-CSF concentration (pg/mL) was assessed by ELISA. Results are given as the mean (± SEM). * P < 0.05; ** P < 0.001; *** P < 0.0001.

Article Snippet: GM-CSF levels were measured in RA SF conditioned media by ELISA assay (Human GM-CSF DuoSet ELISA development kit, R&D Systems).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: Pim1 Serves as a Therapeutic Target for Inflammatory Arthritis via Mitochondrial Metabolism and Th17 Cell Differentiation

doi: 10.34133/research.1137

Figure Lengend Snippet: Pim1 promotes Th17 cell differentiation in vitro. (A) Relative mRNA levels of Pim1 during Th17 cell differentiation ( n = 9). (B) Relative protein levels of pSTAT3, RORγt, and Pim1 during Th17 cell differentiation ( n = 9). (C and D) Frequency of Th17 cells among CD4 + cells after AZD1208 treatment ( n = 9). (E) Relative protein levels of Pim1 among CD4 + T cells overexpressing (OE) vector or Pim1 ( n = 9). (F) Frequency of Th17 cells in CD4 + cells after overexpressing vector or Pim1 ( n = 9). (G) Relative protein levels of RORγt and pSTAT3 after AZD1208 treatment or Pim1 overexpression ( n = 9). (H and I) Relative mRNA levels of Th17-cell-associated pathogenic genes after AZD1208 treatment (H) or Pim1 overexpression (I) ( n = 9). (J and K) Concentration of IL-17A, IL-17F, IL-22, and GM-CSF in the cell supernatant after AZD1208 treatment (J) or Pim1 overexpression (K). (L) Frequencies of Th1, Th2, and Treg cells among CD4 + T cells after treatment with AZD1208 ( n = 9). The data were statistically analyzed via one-way ANOVA, followed by Bonferroni’s post hoc comparisons (A to D) and paired t test (E to L).

Article Snippet: Human IL-17A enzyme-linked immunosorbent assay (ELISA) kit (Elabscience, #E-EL-H5812), human IL-17F ELISA kit (Elabscience, #E-EL-H4193), human IL-22 ELISA kit (Elabscience, #E-EL-H0106), and human GM-CSF ELISA kit (Elabscience, #E-EL-H0081) were used.

Techniques: Cell Differentiation, In Vitro, Plasmid Preparation, Over Expression, Concentration Assay

Journal: Frontiers in Immunology

Article Title: The BCMA-Targeted Fourth-Generation CAR-T Cells Secreting IL-7 and CCL19 for Therapy of Refractory/Recurrent Multiple Myeloma

doi: 10.3389/fimmu.2021.609421

Figure Lengend Snippet: Cytotoxicity analysis of BCMA-hBBz and 7 × 19 CAR-T cells in vitro and in vivo . (A) MM1S-Luc-GFP, U266-Luc-GFP and BCMA-K562 cell lines stably expressing BCMA and luciferase. (B) CAR-T cells and target tumor cells were co-incubated for 4 h at the indicated E:T ratios. Cytotoxicity assay with BCMA-K562 (left), MM1S-Luc-GFP (middle) and U266-Luc-GFP cells as targets (right). Differences between groups were determined using two-way ANOVA. Mean ± SD, **** p < 0.0001. (C) Cytokine release by CAR-T cells in response to multiple myeloma cell lines. CAR-T or mock-T cells were incubated with MM1S-Luc-GFP cells at 1:1 for 24 h, IL2 (left), IFN-γ (middle) and GM-CSF (right) were analyzed by intracellular staining or ELISA. P -value was calculated by two-tailed student t -test. *** P < 0.001. ns, not statistically significant ( P > 0.05). (D) Flow chart of animal experimentation. (E) . On day 0, NSG mice were injected intravenously with 4 × 10 6 BCMA-K562 cells. On day 7, mice received 6 × 10 6 BCMA-7 × 19 CAR-T cells ( n = 3), BCMA-hBBz CAR-T cells ( n = 3) or mock-T cells ( n = 3). Luciferase bioluminescent imaging analysis on days 7, 10, 17, and 24. (F) Average bioluminescent signal for each group in different days [mean radiance (p/s/cm 2 /sr)] ±SD.

Article Snippet: After 24 h, a GM-CSF ELISA kit (BD Biosciences) was used to measure the concentration of GM-CSF in the culture supernatant.

Techniques: In Vitro, In Vivo, Stable Transfection, Expressing, Luciferase, Incubation, Cytotoxicity Assay, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Injection, Imaging