Journal: Acta Neuropathologica Communications

Article Title: Humanized tau antibodies promote tau uptake by human microglia without any increase of inflammation

doi: 10.1186/s40478-020-00948-z

Figure Lengend Snippet: Mouse neonatal primary microglia express microglia-specific markers in culture. a Representative fluorescent photomicrographs showing cell morphology of cultured mouse neonatal primary microglia cells. Green, ionizing calcium-binding adaptor molecule 1 (Iba-1), and blue, DAPI nuclear staining. Scale bars, 50 μm. b Flow cytometry analysis confirmed purity of primary microglia culture. Histogram shows the binding for anti-CD11b compared to the binding for nonspecific IgG. Quantification of four independent microglia cultures confirmed that around 95% of cells were positive for microglia marker CD11b (mean +/− SEM). c Analysis of FcγRs confirmed that almost all microglia express FcγI receptor (CD64), FcγII and FcγIII receptor (CD16/32) (mean +/− SEM)

Article Snippet: Monoclonal mouse antibodies DC8E8 and DC25 [ ], DC51 [ ], DC190 (mapping tau epitope 368–376, Axon Neuroscience SE), rabbit anti-Iba1 (WAKO), blocking antibodies anti-CD16 + CD32 (Abcam) and anti-CD64 (SantaCruz Biotechnologies), rat anti-mouse CD11b Pacific Blue (Biorad), mouse IgG2b isotype control Pacific Blue (Biorad), PE rat anti-mouse CD16/CD32 (BD PharmingenTM), PE mouse IgG2b isotype control (BD PharmingenTM), mouse FcgRIA/CD64a (A594) (R&D systems), and mouse IgG2a isotype control (A594) were used for tau analysis and for mouse microglia.

Techniques: Cell Culture, Binding Assay, Staining, Flow Cytometry, Marker

Journal: Acta Neuropathologica Communications

Article Title: Humanized tau antibodies promote tau uptake by human microglia without any increase of inflammation

doi: 10.1186/s40478-020-00948-z

Figure Lengend Snippet: Human adult primary microglia express specific microglial markers. a Representative fluorescent photomicrographs of human primary microglia culture demonstrating cell morphology and purity of culture; Iba-1 (green), DAPI nuclear staining (blue). Scale bar represents 50 μm. b Flow cytometry analysis for CD11b, CD16, CD32 and CD64 confirmed purity of human microglia cultures. For each marker representative histograms are shown from one human microglia culture and bar graphs with percentage of positive cells from four independent microglia cultures (mean +/− SEM). Histograms and bar graphs show binding and cell positivity for anti-CD11b, −CD16, −CD32 and -CD64, respectively, compared to binding and cell positivity for nonspecific IgG

Article Snippet: Monoclonal mouse antibodies DC8E8 and DC25 [ ], DC51 [ ], DC190 (mapping tau epitope 368–376, Axon Neuroscience SE), rabbit anti-Iba1 (WAKO), blocking antibodies anti-CD16 + CD32 (Abcam) and anti-CD64 (SantaCruz Biotechnologies), rat anti-mouse CD11b Pacific Blue (Biorad), mouse IgG2b isotype control Pacific Blue (Biorad), PE rat anti-mouse CD16/CD32 (BD PharmingenTM), PE mouse IgG2b isotype control (BD PharmingenTM), mouse FcgRIA/CD64a (A594) (R&D systems), and mouse IgG2a isotype control (A594) were used for tau analysis and for mouse microglia.

Techniques: Staining, Flow Cytometry, Marker, Binding Assay

Journal: iScience

Article Title: Rickettsial pathogen inhibits tick cell death through tryptophan metabolite mediated activation of p38 MAP kinase.

doi: 10.1016/j.isci.2022.105730

Figure Lengend Snippet: Figure 3. Anaplasma phagocytophilum downregulates expression of smp30, syntaxin and pld3 mRNA levels in unfed I. scapularis nymphs and tick cells (A–I) Quantitative PCR analysis showing expression of smp30 (A and E), syntaxin (B and F), pld3 (C and G), vps26b (D and H) in unfed uninfected or A. phagocytophilum-infected ticks (A–D) and uninfected or A. phagocytophilum-infected tick cells (E–H) at 48 h p.i. Open circles represent uninfected (UI) and closed circles represent infected (I) ticks or tick cells. In panels (A–D), each circle represents data from samples generated from one tick and in panels (E–H), each circle represents data from tick cell samples generated from one independent culture plate well in a plate. Horizontal lines in the graphs represents mean of the data points. The mRNA levels of these genes were normalized to tick beta-actin mRNA levels. p value from Student’s t test is shown. (I) Immunoblotting analysis showing levels of PLD3 in uninfected (UI) or A. phagocytophilum-infected (I) unfed nymphal ticks. Coomassie blue stained gel image for total protein profile serves as loading control in the immunoblotting analysis. M indicates Precision Plus protein all blue marker from BioRad, USA.

Article Snippet: Ixodes scapularis (larvae, nymph, adult male and females) BEI Resources, NIAID, NIH NR-44115, NR-42510, NR-44116 Chemicals, peptides, and recombinant proteins Potassium sulfate Alfa Aesar Cat # A13975 IMDM media Fisher Scientific Cat # SH3025901 L-glutamine Gibco, USA Cat # 25030-081 FBS VWR, USA Cat # 89510-186 Leibovitz’s L-15 Medium, powder Gibco Cat # 41300-039 Tryptose phosphate broth MP Biomedicals, USA Cat # ICN1682149 Bovine Cholesterol Lipoprotein Concentrate: MP Biomedicals, USA Cat # ICN19147625 Xanthurenic acid (XA) Sigma, USA Cat #D120804-5G Sodium hydroxide VWR Cat # 1310-73-2 DMSO Sigma, USA Cat #D5879-500 ML BIRB796 Tocris, USA Cat # 59-891-0 MTT (Thiazolyl Blue Tetrazolium Bromide) Sigma, USA Cat # MKCR0748 RIPA lysis buffer BioSciences, USA Cat # 786-490 EDTA-free protease inhibitor cocktail Roche Cat # 11836170001 Laemmli sample buffer BioRad Cat # 161-0737 30% Acrylamide BioRad, USA Cat # 1610156 SDS Sigma, USA Cat #L3771-500G APS Columbus Chemical industries, USA Cat # 053500 Tris HCl American Bioanalytical Cat # AB02005-01000 Tris base Sigma, USA Cat #T6066-1 KG Glycine Sigma, USA Cat #G8898-1 KG Hydrochloric acid (HCl) Macron fine chemicals Cat #H613-46 Coomassie Blue BioRad, USA Cat # 1610400 Nitrocellulose membrane Amersham GE Cat # 10600002 BSA Sigma, USA Cat # 9048-46-8 Tween 20 American Bioanalytical Cat # AB02038-00500 Clarity Western ECL substrate BioRad, USA Cat # 1705061 Acetate Buffer ThermoScientific, USA Cat #J60964.AK iTaq Universal SYBR Green Supermix BioRad, USA Cat #L001752 B

Techniques: Expressing, Real-time Polymerase Chain Reaction, Infection, Generated, Western Blot, Staining, Control, Marker

Journal: iScience

Article Title: Rickettsial pathogen inhibits tick cell death through tryptophan metabolite mediated activation of p38 MAP kinase.

doi: 10.1016/j.isci.2022.105730

Figure Lengend Snippet: Figure 6. Anaplasma phagocytophilum upregulates p38 MAPK levels in unfed nymphal ticks and tick cells (A and B) Quantitative PCR analysis showing expression of p38 mapk in unfed uninfected or A. phagocytophilum-infected ticks (A) or tick cells (B). Open circles represent data from uninfected (UI) and closed circles represent data from infected (I) ticks or tick cells. Each circle represents data from one tick (A) or samples collected from one culture plate well in a plate (B). Horizontal lines in the graphs represents mean of the data points. The mRNA levels of these genes were normalized to tick beta-actin mRNA levels. p value from Student’s t test is shown. (C) Immunoblotting analysis showing phosphorylated and total p38 MAPK levels in uninfected (UI) or A. phagocytophilum-infected (I) unfed nymphal ticks. (D) Immunoblotting analysis showing phosphorylated p38 MAPK levels in uninfected (UI) or A. phagocytophilum-infected (I) tick cells. Coomassie blue stained gel image for total protein profile serves as loading control in the immunoblotting analysis. M indicates Precision Plus protein all blue marker from BioRad, USA.

Article Snippet: Ixodes scapularis (larvae, nymph, adult male and females) BEI Resources, NIAID, NIH NR-44115, NR-42510, NR-44116 Chemicals, peptides, and recombinant proteins Potassium sulfate Alfa Aesar Cat # A13975 IMDM media Fisher Scientific Cat # SH3025901 L-glutamine Gibco, USA Cat # 25030-081 FBS VWR, USA Cat # 89510-186 Leibovitz’s L-15 Medium, powder Gibco Cat # 41300-039 Tryptose phosphate broth MP Biomedicals, USA Cat # ICN1682149 Bovine Cholesterol Lipoprotein Concentrate: MP Biomedicals, USA Cat # ICN19147625 Xanthurenic acid (XA) Sigma, USA Cat #D120804-5G Sodium hydroxide VWR Cat # 1310-73-2 DMSO Sigma, USA Cat #D5879-500 ML BIRB796 Tocris, USA Cat # 59-891-0 MTT (Thiazolyl Blue Tetrazolium Bromide) Sigma, USA Cat # MKCR0748 RIPA lysis buffer BioSciences, USA Cat # 786-490 EDTA-free protease inhibitor cocktail Roche Cat # 11836170001 Laemmli sample buffer BioRad Cat # 161-0737 30% Acrylamide BioRad, USA Cat # 1610156 SDS Sigma, USA Cat #L3771-500G APS Columbus Chemical industries, USA Cat # 053500 Tris HCl American Bioanalytical Cat # AB02005-01000 Tris base Sigma, USA Cat #T6066-1 KG Glycine Sigma, USA Cat #G8898-1 KG Hydrochloric acid (HCl) Macron fine chemicals Cat #H613-46 Coomassie Blue BioRad, USA Cat # 1610400 Nitrocellulose membrane Amersham GE Cat # 10600002 BSA Sigma, USA Cat # 9048-46-8 Tween 20 American Bioanalytical Cat # AB02038-00500 Clarity Western ECL substrate BioRad, USA Cat # 1705061 Acetate Buffer ThermoScientific, USA Cat #J60964.AK iTaq Universal SYBR Green Supermix BioRad, USA Cat #L001752 B

Techniques: Real-time Polymerase Chain Reaction, Expressing, Infection, Western Blot, Staining, Control, Marker