Journal: Immunity

Article Title: Single cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes

doi: 10.1016/j.immuni.2018.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-Tmem119 (for mouse staining, Abcam ab209064, 1:500), anti-Tmem119 (for human staining, Sigma, HPA051870 , 1:500), anti-CCL4/MIP-1 beta (for mouse staining, R&D Systems AF-451 1:25), anti-CCL4/MIP-1 beta (for human staining, R&D Systems AF-271, 1:25), anti-HLA-DR (LN3) (Abcam).

Techniques: Staining, Virus, Control, Recombinant, Multiplex Assay, RNAscope, Gene Expression, Software, Plasmid Preparation

Journal: Journal of Inflammation (London, England)

Article Title: Aggregated Hendra virus C-protein activates the NLRP3 inflammasome to induce inflammation

doi: 10.1186/s12950-023-00365-8

Figure Lengend Snippet: HeVc peptide induces IL-1β secretion in murine immortalized macrophages. (A) Beta aggregation score of C-protein amino acids as derived by Tango software alongside a representative image of where the HeVc peptide is taken from the main C-protein. (B) Murine iBMDMs were seeded at 4 × 10 4 per well 20 h prior to priming with 100 ng/ml LPS-B5 for 3 h and then challenged with aggregated HeVc peptide (25–200 µg/ml), or NLRP3 stimulants (6 µM Nigericin or 125 µg/ml Silica) as indicated for an additional 6 h. Supernatants were harvested and assayed for IL-1β by ELISA according to manufacturer’s instructions. Results shown are representative of 3 independent experiments conducted in triplicate, presented as mean ± SD where appropriate, * p < 0.05, ** p < 0.01, *** p < 0.001. Ordinary One-way ANOVA with Dunnett’s test for multiple comparisons

Article Snippet: IL-1β and Caspase-1 were detected using anti-mouse IL-1β detection biotinylated IgG antibody (R&D Systems) and anti-mouse Caspase-1 monoclonal antibody (Adipogen Life Sciences) where indicated.

Techniques: Derivative Assay, Software, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation (London, England)

Article Title: Aggregated Hendra virus C-protein activates the NLRP3 inflammasome to induce inflammation

doi: 10.1186/s12950-023-00365-8

Figure Lengend Snippet: HeVc peptide-induced IL-1β secretion requires phagocytosis, caspase-1 activation and NLRP3 function. Murine iBMDMs were primed with 100 ng/ml LPS-B5 for 2 h before inhibitors of A ) phagocytosis (0.5-2 µM Latrunculin A), B-C ) caspase-1 activity (1–10 µM Ac-YVAD-cmk, 1–5 µM VX695) and D-E ) NLRP3 (1–25 µM MCC950, 87.5-250 µM Probenecid) and were added to cell media for 1 h. Cells were then stimulated with 200 µg/ml of HeVc peptide for 6 h and supernatants were harvested for the detection of IL-1β secretion via ELISA. Results are representative of 3 independent experiments conducted in triplicate and are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, Ordinary One-way ANOVA with Dunnett’s test for multiple comparisons

Article Snippet: IL-1β and Caspase-1 were detected using anti-mouse IL-1β detection biotinylated IgG antibody (R&D Systems) and anti-mouse Caspase-1 monoclonal antibody (Adipogen Life Sciences) where indicated.

Techniques: Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation (London, England)

Article Title: Aggregated Hendra virus C-protein activates the NLRP3 inflammasome to induce inflammation

doi: 10.1186/s12950-023-00365-8

Figure Lengend Snippet: HeVc peptide-induced IL-1β secretion requires an NLRP3, ASC and Caspase-1 inflammasome complex. Murine iBMDMs deficient in either NLRP3, ASC, or Caspase-1 were primed with 100 ng/ml LPS-B5 for 3 h before being stimulated with A ) HeVc peptide (200 µg/ml), B ) Silica (125 µg/ml), C ) Nigericin (6 µM) or D ) poly (dA:dT) for 6 h and supernatants were harvested and assayed for IL-1β secretion by ELISA. Results are pooled and normalized to wildtype response from 3 independent experiments conducted in triplicate and are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, Ordinary One-way ANOVA with Dunnett’s test for multiple comparisons

Article Snippet: IL-1β and Caspase-1 were detected using anti-mouse IL-1β detection biotinylated IgG antibody (R&D Systems) and anti-mouse Caspase-1 monoclonal antibody (Adipogen Life Sciences) where indicated.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation (London, England)

Article Title: Aggregated Hendra virus C-protein activates the NLRP3 inflammasome to induce inflammation

doi: 10.1186/s12950-023-00365-8

Figure Lengend Snippet: HeVc peptide-induces maturation of IL-1β and caspase-1 in human THP-1 macrophages and PBMCs. Human THP-1 cells seeded in a 12-well plate (5 × 10 5 cells per well) were differentiated with 4 µM PMA in media for 16 h before their media was changed and incubated for a further 48 h to differentiate into macrophages. THP-1 derived macrophages were primed with 100 ng/ml LPS-B5 for 3 h in the presence or not of B) MCC950 and A-B) stimulated with 200 µg/ml of HeVc peptide for 6 h. Supernatants were harvested for the detection of IL-1β secretion via ELISA. C-D) THP-1 macrophages were treated with MCC950 or not where indicated and challenged with aggregated HeVc peptide or silica for 6 h respectively. Supernatants were harvested and proteins separated on a 4–12% SDS-PAGE gel before visualization of IL-1β and caspase-1 was conducted by immunoblot. E) hPBMCs from 4 donors were separated from 30 ml blood via density-gradient separation and seeded into a 96-well plate (2.5 × 10 5 cells per well). hPBMCs were primed for 3 h with 50 pg/ml LPS in the presence or not of MCC950 before stimulation with HeVc peptide (200 µg/ml) or Scramble (200 µg/ml) for 6 h. Supernatant was analysed via ELISA for IL-1β secretion. A-B) Results are representative of 3 independent experiments conducted in triplicate and are presented as mean ± SD. E) Results are the mean ± SEM of four independent donors. Ordinary One-way ANOVA with Dunnett’s test for multiple comparisons * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: IL-1β and Caspase-1 were detected using anti-mouse IL-1β detection biotinylated IgG antibody (R&D Systems) and anti-mouse Caspase-1 monoclonal antibody (Adipogen Life Sciences) where indicated.

Techniques: Incubation, Derivative Assay, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Aggregated Hendra virus C-protein activates the NLRP3 inflammasome to induce inflammation

doi: 10.1186/s12950-023-00365-8

Figure Lengend Snippet: HeVc peptide induces inflammation and NLRP3-dependent IL-1β secretion in the lungs of mice. Wildtype C57BL/6 mice were intranasally challenged with 50 µg HeVc peptide in PBS, 50 µg of HeVc peptide and 100 µg (5 mg/kg) MCC950, or PBS alone for 6 h. Bronchoalveolar lavage was harvested. A ) IL-1β and B ) IL-6 secretion was analysed by ELISA of lavage supernatants. Total C ) neutrophil cell numbers as determined by Ly6G + Ly6C − cells, and C) BAL cell counts, determined by flow cytometry. Results are representative of 6 mice per group shown as individual circles. Data presented as mean ± SD. * p < 0.05. Ordinary One-way ANOVA with Dunnett’s test for multiple comparisons

Article Snippet: IL-1β and Caspase-1 were detected using anti-mouse IL-1β detection biotinylated IgG antibody (R&D Systems) and anti-mouse Caspase-1 monoclonal antibody (Adipogen Life Sciences) where indicated.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Infection and Immunity

Article Title: Potential Role of the Chemokine Macrophage Inflammatory Protein 1α in Human and Experimental Schistosomiasis

doi: 10.1128/iai.73.4.2515-2523.2005

Figure Lengend Snippet: FIG. 1. Chemokine levels in the plasma of schistosome-infected patients and control individuals. Individual concentrations are shown with the median of each group expressed as a horizontal line. Patients were classified as intestinal (n 32) or hepatosplenic (n 13) ac- cording to clinical and US examinations. A control group was com- posed of 12 individuals. Plasma samples were processed for elimina- tion of cross-reactivity and used for CCL3 determination by ELISA. The data were analyzed by the Kruskal-Wallis test, followed by Dunn’s comparison test.

Article Snippet: The ELISA kits for mouse IL-4, IL-10, IL-13, gamma interferon (IFN- ), and human CCL3 were obtained from R&D Systems.

Techniques: Clinical Proteomics, Infection, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Infection and Immunity

Article Title: Potential Role of the Chemokine Macrophage Inflammatory Protein 1α in Human and Experimental Schistosomiasis

doi: 10.1128/iai.73.4.2515-2523.2005

Figure Lengend Snippet: FIG. 2. Total number of worms in the portal system and fecundity of S. mansoni harvested from wild-type or CCL3-deficient mice. Val- ues are the means the SEM of worms from perfusates of female mice. (A) The male and female adult mated and unmated worms present in the portal system of infected wild-type (; acute phase, n 6; chronic phase, n 12) or CCL3-deficient (o; acute phase, n 6; chronic phase, n 11) mice were counted after hepatic perfusion at 9 (acute phase) or 14 (chronic phase) weeks after infection. (B) The ratio between the total number or eggs present in all analyzed tissues, i.e., lungs, liver, intestine, and spleen divided by the number of female worms gives the tissue egg retention index for each group. The num- bers of female worms are equal to those used to calculate the averages shown in panel A. #, P 0.01 as determined by the Newman-Keuls test for CCL3-deficient mice compared to wild-type ones at the same phase of infection.

Article Snippet: The ELISA kits for mouse IL-4, IL-10, IL-13, gamma interferon (IFN- ), and human CCL3 were obtained from R&D Systems.

Techniques: Infection

Journal: Infection and Immunity

Article Title: Potential Role of the Chemokine Macrophage Inflammatory Protein 1α in Human and Experimental Schistosomiasis

doi: 10.1128/iai.73.4.2515-2523.2005

Figure Lengend Snippet: FIG. 3. S. mansoni egg burdens in different tissues from wild-type or CCL3-deficient mice. Values are the means the SEM of eggs counted from tissues or feces from female mice. At the day prior to sacrifice, animals were kept in special cages for the collection of their feces. Eggs in the feces (A) of wild-type (; acute phase, n 6; chronic phase, n 12) or CCL3-deficient (o; acute phase, n 6; chronic phase, n 11) mice are expressed per grams. After sacrifice, mice had their livers (B), spleens (C), and intestines (D) excised, weighed, and dissolved in 5% KOH. After the counting of aliquots in a light microscope, the total number of eggs in the tissues was calculated. # and ##, P 0.05 or P 0.01, respectively, as determined by the Newman-Keuls test for MIP-1a/CCL3-deficient mice compared to wild-type mice at the same phase of infection.

Article Snippet: The ELISA kits for mouse IL-4, IL-10, IL-13, gamma interferon (IFN- ), and human CCL3 were obtained from R&D Systems.

Techniques: Light Microscopy, Infection

Journal: Infection and Immunity

Article Title: Potential Role of the Chemokine Macrophage Inflammatory Protein 1α in Human and Experimental Schistosomiasis

doi: 10.1128/iai.73.4.2515-2523.2005

Figure Lengend Snippet: FIG. 4. Blood eosinophils and EPO activity in the livers and intes- tines of wild-type and CCL3-deficient mice. Values are the means the SEM from male mice. At 9 (acute phase) or 14 (chronic phase) weeks after infection, a fragments of the livers (A) and intestines (B) of noninfected () or infected (o) wild-type (acute phase noninfected, n 4; infected, n 6; chronic phase noninfected, n 8; infected, n 12) and CCL3-deficient (acute phase noninfected, n 5; infected, n 4; chronic phase noninfected, n 8; infected, n 12) mice were weighed and processed for quantification of EPO activity. Blood smears were also prepared, fixed, stained with hematoxylin and eosin, and then subjected to differential cell counting. The percentages of eosinophils are shown in panel C. ❋, P 0.001 (in panel A), P 0.05 (in panel B), or P 0.01 (in panel C) as determined by the Newman- Keuls test for infected versus noninfected mice of the same phenotype at the same phase of infection. #, P 0.05 for infected CCL3-deficient compared to infected wild-type mice at the same phase of infection.

Article Snippet: The ELISA kits for mouse IL-4, IL-10, IL-13, gamma interferon (IFN- ), and human CCL3 were obtained from R&D Systems.

Techniques: Activity Assay, Infection, Staining, Cell Counting

Journal: Infection and Immunity

Article Title: Potential Role of the Chemokine Macrophage Inflammatory Protein 1α in Human and Experimental Schistosomiasis

doi: 10.1128/iai.73.4.2515-2523.2005

Figure Lengend Snippet: FIG. 5. Area of the granulomas formed around eggs and hy- droxyproline content in the liver of wild-type and CCL3-deficient mice at the acute and chronic phases of infection. Hydroxyproline content was used as a measure of collagen deposition. Values are the means the SEM of liver samples from male mice at the chronic phase of the infection. (A) At 9 (acute phase) or 14 (chronic phase) weeks after infection, paraffin-fixed liver slices of wild-type ( acute phase, n 8; chronic phase, n 12) or CCL3-deficient (o; acute phase, n 8; chronic phase, n 12) mice were stained with eosin and hematoxylin. Isolated granuloma images were captured and analyzed in the software KS300 2.0 for the measurement of the area. ❋, P 0.01 as determined by the Newman-Keuls test for infected mice at the acute phase com- pared to infected mice of the same phenotype at the chronic phase; #, P 0.01 for CCL3-deficient mice compared to wild-type mice at the same phase of infection. (B) At 14 weeks (chronic phase) after infec- tion, a fragment of the liver of noninfected () or infected (o) wild- type (noninfected, n 4; infected, n 6) and CCL3-deficient (non- infected, n 4; infected, n 7) animals was used for the quantification of hydroxyproline. ❋, P 0.05 for infected mice compared to nonin- fected ones of the same phenotype; #, P 0,05 for infected MIP-1/ CCL3-deficient mice compared to wild-type mice.

Article Snippet: The ELISA kits for mouse IL-4, IL-10, IL-13, gamma interferon (IFN- ), and human CCL3 were obtained from R&D Systems.

Techniques: Infection, Staining, Isolation, Software

Journal: Infection and Immunity

Article Title: Potential Role of the Chemokine Macrophage Inflammatory Protein 1α in Human and Experimental Schistosomiasis

doi: 10.1128/iai.73.4.2515-2523.2005

Figure Lengend Snippet: FIG. 6. Histological sections of the liver of noninfected or infected male wild-type and CCL3-deficient animals at the chronic phase of infection. Images are from Picrus Sirius-stained histological sections representative of the group. (A) Normal architecture of the liver of a noninfected wild-type animal. (B) Normal architecture of the liver of a noninfected CCL3-deficient animal. (C) Appearance of the liver of a wild-type infected mouse at the chronic phase of the infection. Notice the strong granulomatous response formed around the eggs. (D) Appearance of the liver of a CCL3-deficient infected mouse at the chronic phase of the infection. Many of the alterations described in panel C are also visible.

Article Snippet: The ELISA kits for mouse IL-4, IL-10, IL-13, gamma interferon (IFN- ), and human CCL3 were obtained from R&D Systems.

Techniques: Infection, Staining

Journal: Infection and Immunity

Article Title: Potential Role of the Chemokine Macrophage Inflammatory Protein 1α in Human and Experimental Schistosomiasis

doi: 10.1128/iai.73.4.2515-2523.2005

Figure Lengend Snippet: FIG. 7. Cytokine production by MLN cells from male infected and noninfected wild-type and CCL3-deficient mice in response to SEA stimulation at the chronic phase of the infection. Values are the means the SEM of supernatants from four mice. At 14 weeks postinfection, lymph node cells were obtained and stimulated with SEA. The supernatant was collected 48 h later for the quantification of IL-4 (A), IL-10 (B), TNF- (C), and IFN- (D). ❋and ❋❋, P 0.05 or P 0.01, respectively, for infected mice compared to noninfected ones of the same phenotype as determined by the Newman-Keuls test; #, P 0.05 for infected CCL3-deficient compared to infected wild-type mice as determined by unpaired t test. Un., undetectable.

Article Snippet: The ELISA kits for mouse IL-4, IL-10, IL-13, gamma interferon (IFN- ), and human CCL3 were obtained from R&D Systems.

Techniques: Infection

Journal: PloS one

Article Title: CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues.

doi: 10.1371/journal.pone.0066286

Figure Lengend Snippet: Figure 1. Immunohistochemical localization of CCL20 in human IVD tissues and comparison of mean density scores of the positive cells between the three groups. Fifty IVD tissue samples (Group P, n = 20 and Group E, n = 30) from disc degeneration patients and 3 from scoliosis patients were analyzed. Representative results from each group are shown. No distinct CCL20 expression was detected in the control (a). (b) and (c) represent the CCL20 expression analysis of the protrusion and extrusion groups, respectively. Inflammatory reactions on the edges of extruded IVD tissues are shown in (d). The magnifications of the positive cells were shown in the frame of the top right corner of (b), (c) and (d), respectively. (e) and (f) represent the isotype control of (b) and (c), respectively. Magnification, 400x; scale bars, 20 mm. Image-Pro Plus software was used to perform a quantitative analysis of the IHC results. Compared to the normal control, the expression levels of CCL20, shown as the mean densities of the positive cells, were significantly increased in the patient groups (B, *, P,0.01, significantly different from the control, *g, P,0.01, significantly different from Group E). doi:10.1371/journal.pone.0066286.g001

Article Snippet: Endogenous peroxidases in the sections were blocked with 3% hydrogen peroxide, and antigen retrieval was performed by microwaving the sections in EDTA (1:50, ZSGB-Bio, China) at 95uC for 15 min. After blocking the nonspecific proteins with 20% goat serum (ZSGB-Bio, China), goat anti-human CCL20 (5 mg/ml, R&D Systems, Inc., USA) and rabbit anti-human TNF-a (Abcam Inc., USA) primary antibodies were added to the sections, which were incubated overnight at 4uC.

Techniques: Immunohistochemical staining, Comparison, Expressing, Control, Software

Journal: PloS one

Article Title: CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues.

doi: 10.1371/journal.pone.0066286

Figure Lengend Snippet: Figure 4. IL-17A, TNF-a, alone or in combination, can increase the secretion of CCL20 protein from NP cells in a dose- and time- dependent manner. Eight IVD tissue samples from disc degeneration patients were used as the primary cultures. NP cells were stimulated with the indicated concentrations of (a) IL-17A or (b) TNF-a for 48 hours. The cells were treated with the optimal concentrations of (c) IL-17A, TNF-a or both for 0, 24, 48 and 72 hours. The cell-free supernatants were harvested and the expression levels of CCL20 protein were quantified by ELISA. The data are representative of at least three separate experiments, performed in duplicate, and the mean protein levels and standard deviations (SD) are shown. Significant differences were detected by a least significance difference analysis (LSD) (P,0.05). There was statistically significant differences between each concentration of IL-17 when compared to the control; however, statistical significance was verified only for TNF-a concentrations higher than 10 ng/ml (IL-17 treatment: *, P,0.01, vs. control; +, P,0.001, vs. 10 ng/ml; #, p,0.05, vs. 1000 ng/ml; TNF-a treatment: *, p,0.001, vs. control; +, p,0.001, vs. 10 ng/ml). doi:10.1371/journal.pone.0066286.g004

Article Snippet: Endogenous peroxidases in the sections were blocked with 3% hydrogen peroxide, and antigen retrieval was performed by microwaving the sections in EDTA (1:50, ZSGB-Bio, China) at 95uC for 15 min. After blocking the nonspecific proteins with 20% goat serum (ZSGB-Bio, China), goat anti-human CCL20 (5 mg/ml, R&D Systems, Inc., USA) and rabbit anti-human TNF-a (Abcam Inc., USA) primary antibodies were added to the sections, which were incubated overnight at 4uC.

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

Journal: PloS one

Article Title: CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues.

doi: 10.1371/journal.pone.0066286

Figure Lengend Snippet: Figure 5. IL-17A and TNF-a increase CCL20 mRNA expression in NP cells in a dose-dependent manner. Eight IVD tissue samples from disc degeneration patients were used as the primary cultures. The cells were treated with the indicated concentrations of (a) IL-17A or (b)TNF-a for 48 hours. The expression levels of CCL20 mRNA were detected and the CCL20 transcripts were quantified by real-time RT-PCR analyses in all experiments. The data are representative of at least two separate experiments, performed in duplicate, and the mean increases in the mRNA expression were recorded, after which we evaluated the standard deviations (SD). Significant differences were detected by a least significance difference analysis (LSD) (P,0.05). The results correspond with the findings obtained in the ELISA tests. (IL-17 treatment: *, P,0.001, vs. control; +, P,0.05, vs. 10 ng/ml; #, p,0.01, vs. 1000 ng/ml; TNF-a treatment: *, p,0.001, vs. control; +, p,0.01, vs. 10 ng/ml). doi:10.1371/journal.pone.0066286.g005

Article Snippet: Endogenous peroxidases in the sections were blocked with 3% hydrogen peroxide, and antigen retrieval was performed by microwaving the sections in EDTA (1:50, ZSGB-Bio, China) at 95uC for 15 min. After blocking the nonspecific proteins with 20% goat serum (ZSGB-Bio, China), goat anti-human CCL20 (5 mg/ml, R&D Systems, Inc., USA) and rabbit anti-human TNF-a (Abcam Inc., USA) primary antibodies were added to the sections, which were incubated overnight at 4uC.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: iScience

Article Title: Mea6/cTAGE5 cooperates with TRAPPC12 to regulate PTN secretion and white matter development

doi: 10.1016/j.isci.2024.109180

Figure Lengend Snippet:

Article Snippet: Rabbit- anti -TRAPPC2 , Proteintech , Cat# 12484; RRID: AB_2208136.

Techniques: Recombinant, Virus, Software

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: ODN 2088 modulates the release of chemokines by SC astrocytes, in vitro. a Representative chemokine arrays used to detect chemokines in CM of vehicle- and ODN 2088-treated astrocytes. The chemokine arrays were independently repeated twice, showing similar results. Results from a representative experiment are shown. The dots enclosed in rectangular boxes show chemokines whose levels were decreased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to the CM of vehicle-treated astrocytes. The dots enclosed in the oval box show the chemokine whose levels were increased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to CM of vehicle-treated astrocytes. 1: CCL1; 2: CCL9/MIP-1γ; 3: CCL2/MCP-1; 4: CCL20/MIP-3α; 5: CX3CL1. b Densitometric quantification of the signal obtained in the chemokine array using the Image Lab software (Bio-Rad). c Quantification of CCL9 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [** p < 0.01, independent-sample t -test, two-tailed]. The experiment was independently repeated four times, and the mean of 4 experiments ( n = 4) is shown. d Quantification of CCL2 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [* p < 0.05, independent-sample t -test, two-tailed]. The experiment was independently repeated three times, and the mean of 3 experiments ( n = 3) is shown

Article Snippet: In experiments investigating the role of CCL9 in macrophage polarization, recombinant mouse CCL9 (rmCCL9; R&D Systems) was used at the final concentration of 20 pg/ml.

Techniques: In Vitro, Software, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: Astrocyte-derived CCL2 and CCL9 but not CCL1 regulate macrophage polarization, in vitro. a Macrophage cultures were exposed to ODN 2088-treated astrocyte CM (ODN 2088-CM), in the absence or presence of CCL1 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [ p = 0.7228, independent-sample t -test, two-tailed]. b Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL2 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [** p < 0.01, independent-sample t -test, two-tailed]. c Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL9 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [*** p < 0.001, independent-sample t -test, two-tailed]. d Macrophage cultures were exposed to vehicle-treated astrocyte CM (Veh-CM) or ODN 2088-treated astrocyte CM (ODN 2088-CM) for 24 h, with or without (control) addition of rmCCL9 (20 pg/ml). The graph shows the quantification of the F4/80 + /Arg-1 + double-labeled cells expressed as percent of total F4/80 + cells in macrophage cultures [ F (2, 6) = 53.68, p < 0.0001 by one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 by Tukey’s post hoc test]. The experiments were independently repeated twice, yielding similar results. Results from a representative experiment are shown. Results obtained from additional biological repeats of these experiments can be found in Additional file D-G. Data are presented as mean ± SEM

Article Snippet: In experiments investigating the role of CCL9 in macrophage polarization, recombinant mouse CCL9 (rmCCL9; R&D Systems) was used at the final concentration of 20 pg/ml.

Techniques: Derivative Assay, In Vitro, Two Tailed Test, Control, Labeling

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: A scheme summarizing the effects of ODN 2088-treated astrocytes on macrophages. TLR9 antagonism increases the release of CCL1 by astrocytes, which enhances macrophage chemotaxis. In contrast, CCL2 and CCL9 release are decreased in response to ODN 2088. This reduces the negative regulatory effect of CCL2 and CCL9 on M2 macrophage polarization and fosters the M2 phenotype

Article Snippet: In experiments investigating the role of CCL9 in macrophage polarization, recombinant mouse CCL9 (rmCCL9; R&D Systems) was used at the final concentration of 20 pg/ml.

Techniques: Chemotaxis Assay

Journal: Nature Communications

Article Title: The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II

doi: 10.1038/s41467-021-23654-3

Figure Lengend Snippet: a Localization of lysosomes during DC migration in a confined 3D environment. BMDCs were labeled with 1 μM AcidiFluor ORANGE and stimulated with LPS for 2 h. Movement of DCs (left) and AcidiFluor-positive lysosomes (magenta) in response to CCL19 (5 μg/ml) in type I collagen gel (2 mg/ml) in a Zigmond chamber were observed at 1-min intervals by time-lapse video imaging. b , c Localization of LAMP1 + lysosomes ( b ) and Lamtor1 ( c ) in non-polarized and polarized DCs. WT DCs were stained with anti-LAMP1 (green) ( b ), anti-Lamtor1 (red) ( c ), phalloidin (blue), and DAPI (white), and then visualized by confocal microscopy. Polarized and non-polarized cells were identified morphologically. Representative images are shown. Scale bar, 10 μm (left). The percentages of the ROI in the perinuclear and peripheral regions were determined ( n = 30) (right). The perinuclear region was defined as the region within 5 μm of the nuclear membrane, and the peripheral region was defined as the region of the cell outside the perinuclear region. d Co-localization of Lamtor1 and LAMP1 in non-polarized (upper) and polarized (lower) DCs. DCs were stained with anti-Lamtor1 (red), anti-LAMP1 (green), and DAPI (white), and then visualized by confocal microscopy. Representative images are shown. Scale bar, 10 μm. Data are representative of three experiments. e Co-localization of Lamtor1 and phosphorylated MLC. Lamtor1 and phosphorylated MLC were visualized by staining in THP1 expressing Lamtor1-FLAG (red) with anti-p-MLC (green). Representative confocal images of polarized cells (left); scale bar, 10 μm (upper). Intensities of Lamtor1 (red) and p-MLC (green) (lower). Percentage of co-localization of Lamtor1 and p-MLC in body or tail region of polarized cells ( n = 25 cells) (right). Data are representative of three experiments. Statistical analyses were performed by two-sided Steel–Dwass test ( b , c ) or Mann–Whitney U test ( e ) [median; 25th and 75th percentiles; and minimum and maximum of a population excluding outliers; *p < 0.0001].

Article Snippet: Reagents were obtained from the indicated suppliers: LPS, PMA, rapamycin, FITC-isomer I, fibronectin OVA (Grade VI) CFA (Sigma); 2,4-DNFB (Tokyo Chemical Industry); collagenase D (Roche); type I collagen (BD Biosciences); calcein-AM, CFSE (carboxyfluorescein diacetate succinimidyl ester), phalloidin-546 (Invitrogen); AcidiFluor-ORANGE (GORYO Chemical); Torin1 (Selleck); U0216 (Calbiochem); blebbistatin (Cayman Chemical); recombinant mouse CCL19, mouse CCL21, human MCP-1 (R & D Systems); mouse GM-CSF (Wako); and Evans blue (Nacalai Tesque).

Techniques: Migration, Labeling, Imaging, Staining, Confocal Microscopy, Membrane, Expressing, MANN-WHITNEY

Journal: Nature Communications

Article Title: The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II

doi: 10.1038/s41467-021-23654-3

Figure Lengend Snippet: a Chemotaxis of WT (white bar) and Lamtor1 −/− (black bar) BMDCs toward the indicated concentrations of CCL19 (upper) or CCL21 (lower) in a Transwell assay system (pore size, 5 μm). Data are representative of three experiments. b – f Motility of DCs in response to CCL19 in 3D collagen matrices. Movement of WT (upper) and Lamtor1 –/– (lower) BMDCs in response to CCL19 (5 μg/ml) in type I collagen gels (2 mg/ml) in a Zigmond chamber were observed at 1-min intervals by time-lapse video imaging. Velocities of DCs were determined using the ImageJ manual tracking software. Consecutive images of DC locomotion: time (h:min) is shown above each panel. Scale bar, 30 μm ( b ). Mean velocity of the leading edge ( c ) and uropod ( d ), the maximum length of a single cell ( e ), and percentage of the immotile period of the uropod ( f ) ( n = 40 WT cells and n = 40 KO cells). g MLC phosphorylation in WT and Lamtor1 −/− BMDCs. MLC phosphorylation was evaluated by western blotting with anti-p-MLC antibody (upper). Data are representative of three experiments. Protein concentration in SDS–PAGE gel bands was determined using ImageJ, and statistical analysis was performed (lower) ( n = 3). h Localization of LAMP1 + lysosomes in non-polarized and polarized DCs. Lamtor1 −/− DCs were stained with anti-LAMP1 (green), phalloidin (blue), and DAPI (white), and visualized by confocal microscopy. Representative images of a non-polarized cell (left) and a polarized cell (right). Scale bar, 10 μm (left). Percentage of the region of interest (ROI) of perinuclear and peripheral regions of non-polarized cells and polarized cells ( n = 30) (right). i Lamp1 protein level of WT and Lamtor1 −/− BMDCs. The amount of Lamp1 protein was evaluated by western blotting with anti-Lamp1 antibody (upper). Data are representative of three experiments. The protein concentration in SDS–PAGE gel bands was determined using ImageJ and statistical analysis was performed (lower) ( n = 3). Statistical analyses were performed by two-sided Student’s t -test ( a , g , i ) [means ± s.d.; *p < 0.05, ** p < 0.01, ***p < 0.005] or two-sided Mann–Whitney U test ( c – f , h ) [median; 25th and 75th percentiles; and minimum and maximum of a population excluding outliers; *p < 0.001, ** p < 0.0001; NS, not statistically significant].

Article Snippet: Reagents were obtained from the indicated suppliers: LPS, PMA, rapamycin, FITC-isomer I, fibronectin OVA (Grade VI) CFA (Sigma); 2,4-DNFB (Tokyo Chemical Industry); collagenase D (Roche); type I collagen (BD Biosciences); calcein-AM, CFSE (carboxyfluorescein diacetate succinimidyl ester), phalloidin-546 (Invitrogen); AcidiFluor-ORANGE (GORYO Chemical); Torin1 (Selleck); U0216 (Calbiochem); blebbistatin (Cayman Chemical); recombinant mouse CCL19, mouse CCL21, human MCP-1 (R & D Systems); mouse GM-CSF (Wako); and Evans blue (Nacalai Tesque).

Techniques: Chemotaxis Assay, Transwell Assay, Pore Size, Imaging, Software, Phospho-proteomics, Western Blot, Protein Concentration, SDS Page, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: Nature Communications

Article Title: The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II

doi: 10.1038/s41467-021-23654-3

Figure Lengend Snippet: a , b Chemotaxis of DCs treated with mTORC1 inhibitors in response to CCL19. Chemotaxis of WT BMDCs in response to CCL19 (500 ng/ml) in the presence of the indicated concentration of rapamycin ( a ) and torin1 ( b ), as determined by Transwell assay (pore size, 5 μm) (upper). Data are representative of three experiments (mean ± s.d.). Phosphorylation of S6K following treatment with rapamycin ( a ) and Torin1 ( b ) was determined by western blotting (lower). c DC migration in 3D collagen matrices in the presence of rapamycin. Movement of WT BMDCs in response to CCL19 (5 μg/ml) in type I collagen gel (2 mg/ml) containing 10 nM rapamycin was observed by time-lapse video imaging, and the velocities of the uropod (upper) and the percentage of the immotile period of the uropod (lower) were analyzed using the ImageJ manual tracking software ( n = 40). d MLC phosphorylation of DCs treated with rapamycin. MLC phosphorylation and S6K phosphorylation in the presence of 10 nM rapamycin was evaluated by western blotting with anti-p-MLC and anti-p-S6K antibody, respectively (left). Data are representative of three experiments. The concentration of p-MLC in SDS-PAGE gel bands was determined using ImageJ, and statistical analysis was performed (right) ( n = 3). e Chemotaxis of U0126-treated DCs in response to CCL19. Chemotaxis of WT BMDCs in response to CCL19 (500 ng/ml) in the presence of the indicated concentration of U0126, a MEK inhibitor, as determined by Transwell assay (pore size, 5 μm) (upper). Data are representative of three experiments (mean ± s.d.). f MLC phosphorylation of DCs treated with U0126. MLC and ERK phosphorylation in the presence of 20 μM U0126 were determined by western blotting with anti-p-MLC and anti-p-ERK antibody (left). Data are representative of three experiments. The concentration of p-MLC in SDS–PAGE gel bands was determined using ImageJ, and statistical analysis was performed (right) ( n = 3). Statistical analysis was performed by two-sided Student’s t -test ( a , b , d – f ) [means ± s.d.], and two-sided Mann–Whitney U test ( c ) [median; 25th and 75th percentiles; and minimum and a maximum of a population; NS not statistically significant].

Article Snippet: Reagents were obtained from the indicated suppliers: LPS, PMA, rapamycin, FITC-isomer I, fibronectin OVA (Grade VI) CFA (Sigma); 2,4-DNFB (Tokyo Chemical Industry); collagenase D (Roche); type I collagen (BD Biosciences); calcein-AM, CFSE (carboxyfluorescein diacetate succinimidyl ester), phalloidin-546 (Invitrogen); AcidiFluor-ORANGE (GORYO Chemical); Torin1 (Selleck); U0216 (Calbiochem); blebbistatin (Cayman Chemical); recombinant mouse CCL19, mouse CCL21, human MCP-1 (R & D Systems); mouse GM-CSF (Wako); and Evans blue (Nacalai Tesque).

Techniques: Chemotaxis Assay, Concentration Assay, Transwell Assay, Pore Size, Phospho-proteomics, Western Blot, Migration, Imaging, Software, SDS Page, MANN-WHITNEY

Journal: Blood

Article Title: CX3CL1/fractalkine is released from apoptotic lymphocytes to stimulate macrophage chemotaxis.

doi: 10.1182/blood-2008-06-162404

Figure Lengend Snippet: Figure 1. Production of macrophage chemotactic factors by apoptotic BL cells. (A) Chemotaxis of HMDM to EBV-positive (Mutu-BL) and EBV-negative (BL2) lines undergoing spontaneous (f) or UV-induced apoptosis ( ). Bcl-2 transfectants that are protected from apoptosis ( ) are shown for comparison. Chemotaxis is shown as fold increase above that of background (medium alone), which was set to 1. CCL5 (100 ng/mL) is included as a positive control. Levels of apoptosis (assessed using annexin V) for Mutu-BL: 23% (BL), 15% (Bcl-2), 89% (UV); for BL2: 44% (BL), 18% (Bcl-2), 99% (UV). Data shown are means plus or minus SEM of replicate high-power fields. Experiment shown is representative of 3. Student t test (background vs BL cells): **P .005, ***P .001. (B) Chemotaxis of human monocyte/macrophage cell line MonoMac6 to Mutu-BL cells undergoing spontaneous or UV-induced apoptosis. Student t test (background vs BL cells): ***P .001. (C) Kinetics of macrophage chemoattractant release from BL cells undergoing apoptosis. Mutu-BL cells were induced into apoptosis by UV irradiation and assayed at the indicated times. Chemotaxis of HMDM toward apoptotic BL cells (f, mean SD) and apoptosis (assessed using annexin V, ) were assessed in parallel. Experiment is representative of 2 identical. (D) Presence of macrophage chemoattractant activity in cell-free supernatants of BL cells undergoing apoptosis. Chemotaxis of HMDM to UV-induced Mutu-BL cells (f) and to cell-free supernatant (S/N, u) from the same cells. Cells were 94% apoptotic (assessed using annexin V) in this experiment. Student t test (cells vs S/N): P .2 (ns indicates not significant). (E) Blockade of macrophage chemotaxis to apoptotic BL cells by PTX. Chemotaxis to UV-induced Mutu-BL cells of HMDM (u) or HMDM pretreated with PTX (100 ng/mL) for 12 hours before chemotaxis assay (f). Student t test (control vs PTX-treated HMDM): ***P .001. Levels of apoptosis (assessed using annexin V) were 67% (Mutu), 85% (Mutu UV). Note there was no loss in viability of macrophages after PTX treatment (91% viable PTX-treated macrophages at the end of the chemotaxis assay vs 90% for control macrophages in the experiment shown). (F) Inhibition of macrophage chemotaxis to apoptotic BL cells by the viral chemokine antagonist, vMIPII. Chemotaxis of HMDM to UV-induced Mutu-BL cells in the absence (u) or presence (f) of vMIPII (60 ng/mL). Student t test (control vs vMIPII-treated HMDM): ***P .001. Levels of apoptosis (assessed using annexin V) were 51% (Mutu), 88% (Mutu UV). Macrophage viability ( 90%) was unaffected by vMIPII.

Article Snippet: In other experiments, neutralizing anti-FKN monoclonal antibody (50 g/mL, clone 51637.11; R&D Systems) or viral macrophage inflammatory protein II (vMIPII; 60 ng/mL; R&D Systems) were added to the lower wells of the chambers; recombinant human FKN (100 ng/mL chemokine domain; R&D Systems) was included in the upper well in other experiments.

Techniques: Chemotaxis Assay, Comparison, Positive Control, Irradiation, Activity Assay, Control, Inhibition

Journal: Blood

Article Title: CX3CL1/fractalkine is released from apoptotic lymphocytes to stimulate macrophage chemotaxis.

doi: 10.1182/blood-2008-06-162404

Figure Lengend Snippet: Figure 2. Expression of FKN and its cognate recep- tor by BL cells vs mononuclear phagocytes. (A) FKN RT-PCR products amplified from Mutu-BL cells (BL), Bcl-2 transfectants (BLBcl-2), and UV-induced Mutu-BL cells (BLUV). -actin products are shown for comparison. (B) Expression of FKN on the surface of BL cells. BL2 cells were labeled by indirect immunofluo- rescence using anti-FKN mAb (clone 81513). Flow cytometric histograms of surface FKN (dark gray) compared with background immunostaining (light gray) are shown. (C) Immunoblots showing FKN expression in lysates of BL2 cell lines and BL tumors. SDS-PAGE (30 g of protein per lane) was performed on lysates from BL2 cells (left panels) and 3 separate BL2 tumors grown in SCID mice (right panels). Proteins were transferred to Hybond P membrane and immunoblotted using anti-FKN antibody (clone 81513). Blots were stripped and reprobed for -actin to check for loading variability. (D) Expression of FKN in tumor tissue in situ. Immunohistochemical staining was performed on ar- chive paraffin sections of BL tumor tissue. Diaminoben- zidine (DAB) chromogen product (brown) represents FKN staining (hematoxylin counterstain). Primary anti- FKN mAb (clone 81513, top panel); isotype control mAb (mouse IgG1, bottom panel). Arrow indicates example of starry-sky macrophage. Bar represents 52 m. Stained tissue was analyzed using a Zeiss Axioskop 2 microscope (Zeiss, Welwyn Garden City, United Kingdom) at 20°C with objective lens Plan- NEOFLUAR 20 (numerical aperture 0.50) and histo- mount medium (RaymondA. Lamb, Eastbourne, United Kingdom), and images were taken using Jenoptik ProgRes C14 camera (Jenoptik, Jena, Germany) with Openlab 4.0.2 software (Improvision/PerkinElmer, Coventry, United Kingdom). (E) FKN receptor, CX3CR1 RT-PCR products amplified from HMDM (Mac), and the monocyte/macrophage cell lines THP-1 and Mono- Mac6 (MM6). -actin products are shown for compari- son. (F) Expression of FKN receptor, CX3CR1 on the surface of human monocytes, macrophages, and mono- cyte/macrophage cell lines THP-1 and MM6 but not BL cells (BL2 and Mutu-BL). Viable cells were labeled by indirect immunofluorescence using anti-CX3CR1 poly- clonal antibody primary and PE-conjugated secondary. Flow cytometric histograms of surface FKN (dark gray) compared with background immunostaining (light gray) are shown.

Article Snippet: In other experiments, neutralizing anti-FKN monoclonal antibody (50 g/mL, clone 51637.11; R&D Systems) or viral macrophage inflammatory protein II (vMIPII; 60 ng/mL; R&D Systems) were added to the lower wells of the chambers; recombinant human FKN (100 ng/mL chemokine domain; R&D Systems) was included in the upper well in other experiments.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison, Labeling, Immunostaining, Western Blot, SDS Page, Membrane, In Situ, Immunohistochemical staining, Staining, Control, Microscopy, Software

Journal: Blood

Article Title: CX3CL1/fractalkine is released from apoptotic lymphocytes to stimulate macrophage chemotaxis.

doi: 10.1182/blood-2008-06-162404

Figure Lengend Snippet: Figure 3. Requirement for FKN and its receptor, CX3CR1, in chemotaxis of macrophages to apopto- tic cells. (A) Effect of anti-FKN neutralization on the activity of chemoattractants released from apoptotic cells. Chemotaxis of HMDM to Mutu-BL cells undergo- ing spontaneous or UV-induced apoptosis in the pres- ence (f) or absence (u) of neutralizing anti-FKN antibody (50 g/mL) in the lower chamber. Chemotaxis is shown as fold increase above that of background (medium alone), which was set to 1. CCL5 (100 ng/mL) is included as a positive control. Data shown are mean plus or minus SEM of replicate high-power fields. Experiment shown is representative of 4 similar. Stu- dent t test (control vs anti-FKN treatment): ***P .001. (B) Effect of exogenous FKN on macrophage chemo- taxis to apoptotic cells. Chemotaxis of HMDM to Mutu-BL cells undergoing spontaneous or UV-induced apoptosis in the presence (f) or absence (u) of exogenous recombinant human FKN (100 ng/mL) in the upper chamber. Experiment shown is representa- tive of 6 similar. Student t test (control vs FKN treat- ment): ***P .001. (C) Absence of FKN receptor CX3CR1 partially prevents macrophage chemotaxis to apoptotic cells. Chemotaxis of bone marrow–derived macrophages from wild-type (u) or CX3CR1/ mice (f) toward cell-free supernatants of Mutu-BL cells at the indicated times after UV irradiation. C5a (6.25 ng/mL) is included as a positive control. Experiment shown is representative of 6. Student’s t test (control vs anti-FKN cells): ***P .001.

Article Snippet: In other experiments, neutralizing anti-FKN monoclonal antibody (50 g/mL, clone 51637.11; R&D Systems) or viral macrophage inflammatory protein II (vMIPII; 60 ng/mL; R&D Systems) were added to the lower wells of the chambers; recombinant human FKN (100 ng/mL chemokine domain; R&D Systems) was included in the upper well in other experiments.

Techniques: Chemotaxis Assay, Neutralization, Activity Assay, Positive Control, Control, Recombinant, Derivative Assay, Irradiation

Journal: Blood

Article Title: CX3CL1/fractalkine is released from apoptotic lymphocytes to stimulate macrophage chemotaxis.

doi: 10.1182/blood-2008-06-162404

Figure Lengend Snippet: Figure 5. Role of FKN in apoptotic germinal center B cells and other lymphocytes. (A) Expression of FKN in normal lymphoid tissue in situ. Immunohisto- chemical staining was performed on archive paraffin sections of human tonsil. DAB chromogen product (brown) represents FKN staining (hematoxylin counter- stain). Primary anti-FKN mAb (clone 81513); isotype control mAb (mouse IgG1) produced negative results, not shown. GC indicates germinal center of lymphoid follicle. Arrows indicate examples of tingible body mac- rophages. Bar represents 52 m (left panel); bar represents 27 m (right panel). Stained tissue was analyzed using a Zeiss Axioskop 2 microscope (Zeiss) at 20°C with objective lenses Plan-NEOFLUAR 20 (numerical aperture 0.50) and Plan-NEOFLUAR 40 magnification (numerical aperture 0.75) and histo- mount medium (Raymond A. Lamb), and images were taken using Jenoptik ProgRes C14 camera (Jenoplite) with Openlab 4.0.2 software (Improvision/PerkinElmer). (B) Release of FKN by murine germinal center B cells. Immunoblots using rabbit antimouse FKN polyclonal Ab (Torrey Pines Biolabs, East Orange, NJ) of cell-free supernatants (equal volumes of TCA/acetone-precipi- tated supernatants per lane) showing changes in FKN in purified germinal center B cells (GC) and follicular B cells (fol) from mouse spleens cultured in vitro for 2 or 18 hours. (C) Effects of loss of CX3CR1 on macro- phage migration and of clearance of apoptotic cells in lymphoid tissue in vivo. Quantitative immunohistochemi- cal analyses were performed on splenic follicles and extrafollicular areas of mice (wt, CX3CR1/, and CD14/) immunized with SRBCs. Numbers of macro- phages and apoptotic cells in situ were determined by counting CD68 and TUNEL cells per millimeter- squared, respectively. CD14/ mice were used as positive control animals that are known to show defec- tive apoptotic cell clearance in vivo. Significant reduc- tion in numbers of macrophages in follicles was ob- served in CX3CR1/ mice, but apoptotic cells did not persist in these animals. Results shown are mean plus or minus SEM (n 3-6 spleens); ANOVA, wt vs CX3CR1/, ***P .009; wt vs CD14/, **P .018. (D) Modulation and release of FKN during apoptosis of activated primary lymphocytes (CD19-depleted). Flow cytometric analyses showing loss of surface FKN labeling of CD19-depleted lymphocytes after induction of apoptosis. Cells were preactivated by culture with 2 g/mL concanavalin A to obtain surface FKN labeling (freshly isolated cells were surface FKN-negative, data not shown). Cells were labeled by indirect immunofluo- rescence using anti-FKN mAb (clone 81513) at the indicated times after induction of apoptosis by staurospor- ine treatment (1 M). Histograms of viable zone cells are as follows: black represents time 0; white, 1 hour; coarse stippling, 2 hours; fine stippling, 4.5 hours after treatment; pale gray, isotype control immunostaining. Percentages of “FKN-bright” cells lying within the indicated gate are shown. Levels of apoptosis (assessed using annexin V) were 8%, 17%, 18%, and 28% at 0, 1, 2, and 4.5 hours after treatment, respectively. Representative experiment from 3.

Article Snippet: In other experiments, neutralizing anti-FKN monoclonal antibody (50 g/mL, clone 51637.11; R&D Systems) or viral macrophage inflammatory protein II (vMIPII; 60 ng/mL; R&D Systems) were added to the lower wells of the chambers; recombinant human FKN (100 ng/mL chemokine domain; R&D Systems) was included in the upper well in other experiments.

Techniques: Expressing, In Situ, Staining, Control, Produced, Microscopy, Software, Western Blot, Cell Culture, In Vitro, Migration, In Vivo, TUNEL Assay, Positive Control, Labeling, Isolation, Immunostaining