Journal: Nature Protocols

Article Title: Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope

doi: 10.1038/nprot.2014.155

Figure Lengend Snippet: Figure 3 | The components of the mmTIRF system and their assembly. (a) A diagram of the mmTIRF system (Mad City Labs) showing how each of the components interface. The system is designed around an objective holder fixed to a metal plate. A Nano-View/M 200-3 embedded-style xy-micrometer stage is then attached to this same plate. The nanopositioner itself is recessed into the micropositioner. A z-positioner with a dampening mass and a clamp to hold the U-shaped microscope slide holder is secured to the top plate of the nanopositioner. (b) The assembled micromirror TIRF system showing the objective platform, stages, legs and micromirror platform assemblies.

Article Snippet: Z488RDC, Z532RDC and ZT594RDC) Doublet lens for final focusing/steering lens (L3; Newport Corporation, cat. no. PAC067AR.14) Optical support rod for mounting He-Ne laser (Newport Corporation, cat. no. 70) Rack-and-pinion rod clamp part of EM-CCD camera mount (Newport Corporation, cat. no. 370-RC) Cylindrical laser mount (Newport Corporation, cat. no. ULM-TILT) Stainless steel optical posts for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, TR series posts) Post holders for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, PH series post holders) Post-holder bases (Thorlabs BA series) Kinematic cage mounts for mounting xy-translation mounts containing doublet lenses or objectives (Thorlabs, cat. no. KC1-T (×9)) xy-translation mounts for housing doublet lenses and objectives (Thorlabs, cat. no. LM1XY (9×)) Linear stage for fine translation mounted doublet lenses (Newport, cat. no. 423 (5×)) Rotation stage for mounting wave plates (Newport, cat. no. RSP-1T (6); Melles Griot, cat. no. 1100-10 (2×)) Kinematic platform mounts for mounting broadband polarizing cubes (Thorlabs, cat. no. KM100B (4×)) Clamping arm for mounting broadband polarizing cubes (Thorlabs, cat. no. PM3 (4×)) Precision kinematic mirror mounts (Newport Corporation, 1-inch diameter, cat. no. U100-A3K (6×)) Kinematic prism mount for mounting dichroic mirror clamps (Thorlabs, cat. no. KM100PM (3×)) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Plate holder for mounting dichroic mirror (Thorlabs, cat. no. FP01 (3×)) Post-mounted iris diaphragms (I2; Thorlabs, cat. no. ID25 (3×)) Polarizing sheet plate (Thorlabs, cat. no. LPVIS100) Shearing interferometer (Thorlabs, cat. no. SI100) Objective and micromirror platforms Olympus 60× 1.45-NA PlanApo Objective (MO) mmTIRF system (Mad City Labs) containing the following components: shelf braces (cat. no. 629065B (4×)); SM1-threaded ports/side plate (cat. no. 629075B (2×)); RM21 legs (cat. no. 628415 (4×)); sample holder (made from part nos.

Techniques: Microscopy

Journal: Nature Protocols

Article Title: Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope

doi: 10.1038/nprot.2014.155

Figure Lengend Snippet: Figure 5 | A diagram of the position of the components of the microscope on the optical table.

Article Snippet: Z488RDC, Z532RDC and ZT594RDC) Doublet lens for final focusing/steering lens (L3; Newport Corporation, cat. no. PAC067AR.14) Optical support rod for mounting He-Ne laser (Newport Corporation, cat. no. 70) Rack-and-pinion rod clamp part of EM-CCD camera mount (Newport Corporation, cat. no. 370-RC) Cylindrical laser mount (Newport Corporation, cat. no. ULM-TILT) Stainless steel optical posts for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, TR series posts) Post holders for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, PH series post holders) Post-holder bases (Thorlabs BA series) Kinematic cage mounts for mounting xy-translation mounts containing doublet lenses or objectives (Thorlabs, cat. no. KC1-T (×9)) xy-translation mounts for housing doublet lenses and objectives (Thorlabs, cat. no. LM1XY (9×)) Linear stage for fine translation mounted doublet lenses (Newport, cat. no. 423 (5×)) Rotation stage for mounting wave plates (Newport, cat. no. RSP-1T (6); Melles Griot, cat. no. 1100-10 (2×)) Kinematic platform mounts for mounting broadband polarizing cubes (Thorlabs, cat. no. KM100B (4×)) Clamping arm for mounting broadband polarizing cubes (Thorlabs, cat. no. PM3 (4×)) Precision kinematic mirror mounts (Newport Corporation, 1-inch diameter, cat. no. U100-A3K (6×)) Kinematic prism mount for mounting dichroic mirror clamps (Thorlabs, cat. no. KM100PM (3×)) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Plate holder for mounting dichroic mirror (Thorlabs, cat. no. FP01 (3×)) Post-mounted iris diaphragms (I2; Thorlabs, cat. no. ID25 (3×)) Polarizing sheet plate (Thorlabs, cat. no. LPVIS100) Shearing interferometer (Thorlabs, cat. no. SI100) Objective and micromirror platforms Olympus 60× 1.45-NA PlanApo Objective (MO) mmTIRF system (Mad City Labs) containing the following components: shelf braces (cat. no. 629065B (4×)); SM1-threaded ports/side plate (cat. no. 629075B (2×)); RM21 legs (cat. no. 628415 (4×)); sample holder (made from part nos.

Techniques: Microscopy

Journal: Nature Protocols

Article Title: Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope

doi: 10.1038/nprot.2014.155

Figure Lengend Snippet: Figure 7 | The components and layout of the emission path optics. (a) Location of the micromirrors, iris and the 45° mirror under the microscope objective. (b) Mounting of the EM-CCD camera. (c) The dual-view optics as seen looking toward the EM-CCD camera. (d) The dual-view optics as viewed looking toward the microscope objective.

Article Snippet: Z488RDC, Z532RDC and ZT594RDC) Doublet lens for final focusing/steering lens (L3; Newport Corporation, cat. no. PAC067AR.14) Optical support rod for mounting He-Ne laser (Newport Corporation, cat. no. 70) Rack-and-pinion rod clamp part of EM-CCD camera mount (Newport Corporation, cat. no. 370-RC) Cylindrical laser mount (Newport Corporation, cat. no. ULM-TILT) Stainless steel optical posts for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, TR series posts) Post holders for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, PH series post holders) Post-holder bases (Thorlabs BA series) Kinematic cage mounts for mounting xy-translation mounts containing doublet lenses or objectives (Thorlabs, cat. no. KC1-T (×9)) xy-translation mounts for housing doublet lenses and objectives (Thorlabs, cat. no. LM1XY (9×)) Linear stage for fine translation mounted doublet lenses (Newport, cat. no. 423 (5×)) Rotation stage for mounting wave plates (Newport, cat. no. RSP-1T (6); Melles Griot, cat. no. 1100-10 (2×)) Kinematic platform mounts for mounting broadband polarizing cubes (Thorlabs, cat. no. KM100B (4×)) Clamping arm for mounting broadband polarizing cubes (Thorlabs, cat. no. PM3 (4×)) Precision kinematic mirror mounts (Newport Corporation, 1-inch diameter, cat. no. U100-A3K (6×)) Kinematic prism mount for mounting dichroic mirror clamps (Thorlabs, cat. no. KM100PM (3×)) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Plate holder for mounting dichroic mirror (Thorlabs, cat. no. FP01 (3×)) Post-mounted iris diaphragms (I2; Thorlabs, cat. no. ID25 (3×)) Polarizing sheet plate (Thorlabs, cat. no. LPVIS100) Shearing interferometer (Thorlabs, cat. no. SI100) Objective and micromirror platforms Olympus 60× 1.45-NA PlanApo Objective (MO) mmTIRF system (Mad City Labs) containing the following components: shelf braces (cat. no. 629065B (4×)); SM1-threaded ports/side plate (cat. no. 629075B (2×)); RM21 legs (cat. no. 628415 (4×)); sample holder (made from part nos.

Techniques: Microscopy

Journal: Nature Communications

Article Title: Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice

doi: 10.1038/s41467-022-33932-3

Figure Lengend Snippet: a C4a and C4b restore the astrocytic engulfment of microglial debris in vitro in serum-free culture medium. b Quantifications of the phagocytic influence by complement supplementation and preopsonization in serum-free culture medium. N = 11 independent biological replicates of each group. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). c Scheme of in vivo microglial depletion and time points for analysis. d Reanalysis of RNA-seq data from whole-brain homogenate (GSE108269 ) showing that Gfap and C4b are upregulated and C1qa is downregulated during microglial ablation, whereas C2 , C3 and C4a remain at low levels and are unaffected. N = 5 mice at D0 and N = 4 mice at D2 to D21. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). e qPCR further confirmed the upregulation of C4b in sorted astrocytes upon microglial depletion. N = 5 in each group. Two-tailed independent t test. f Scheme of the in vivo examination of astrocytic engulfment using AAV PHP.eB-based astrocyte labeling and microglial depletion. g Confocal orthogonal colocalization and 3D reconstruction show that C4b −/− impairs the astrocytic engulfment of microglial debris under physiological condition (D21) and upon CSF1R inhibition (D23). h Quantification of microglial debris engulfment by astrocytes. N = 7 (D21) and 8 (D23) WT mice, and N = 3 (D21) and 5 (D23) C4b −/− mice. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). PLX5622 PLX5622-formulated AIN-76A diet, CD control AIN-76A diet, IV intravenous, MFI mean fluorescence intensity, Ctx cortex, Hipp hippocampus, OB olfactory bulb. Data are presented as mean ± SD. The source data are provided as a Source Data file.

Article Snippet: Recombinant mouse complement component C2 protein CF (C2) was acquired from R&D Systems (Cat#: 6725-SE-010).

Techniques: In Vitro, In Vivo, RNA Sequencing, Two Tailed Test, Labeling, Inhibition, Control, Fluorescence

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 2. In vitro function of islets pre-cultured with exogenous complement component C3a. Insulin release at 2 mmol/L and 20 mmol/L glucose of 1020 replicates of three mouse islets per Eppendorf tube: (A) pre-cultured alone, with 10 nmol/L C3a alone or with 100 nmol/L C3a alone, for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a; (B) pre-cultured alone, with 5 nmol/L ANXA1 alone or with 10 nmol/L C3a alone for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a or ANXA1; (C) pre-cul- tured alone, with 5 nmol/L ANXA1 alone or with a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a for 48 h followed by subse- quent GSIS assays in the absence of exogenous C3a and/or ANXA1, P < 0.05 versus islets pre-cultured alone at the same glucose concentration. (D) Protection from cytokine-induced apoptosis following a 48 h pre-culture with 5 nmol/L ANXA1-alone, 10 nmol/L C3a alone or a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a, and the subsequent presence of specified MSC biotherapeutics during the final 20 h cytokine incubation. Eight to 12 replicates of five islets per well in each culture group assayed, *P < 0.05 versus islets pre-cul- tured alone in the presence of cytokines for the final 20 h of the 3-day culture period. The P values (AD) were calculated using two-way ANOVA Bonferroni’s post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vitro, Cell Culture, Concentration Assay, Incubation

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 3. Pre-culturing islets with a cocktail of MSC secretory factors ensures sustained improvements to islet insulin secretory function and protection from cytokine-induced apoptosis. (A, B) Insulin release at 2 and 20 mmol/L glucose of 30 replicates of three mouse islets per Eppendorf tube, pre-cultured alone, with 5 nmol/L ANXA1 alone, with 5 nmol/L ANXA1 and 10 nmol/L SDF-1, with 5 nmol/L ANXA1 and 10 nmol/L C3a, or with a cocktail of 5 nmol/L ANXA1, 10 nmol/L SDF-1 and 10 nmol/L C3a, for 48 h, before removal of the MSC- derived biotherapeutics for 1 day (A) or 3 days (B), *P < 0.05 and **P < 0.01 versus islets cultured alone at the same glucose concentration. (C, D) Protection of islets from cytokine-induced apoptosis after pre-culture with MSC-derived biotherapeutics alone, in dual combination or a cocktail of all three factors (as of legend) for 48 h, before removal of the MSC-derived biotherapeutics for 1 day (C) or 3 days (D), 8 to 12 replicates of five islets per well were assayed, *P < 0.05 and **P < 0.01 versus islets cultured alone with cytokines, +P < 0.05 vs. islets cul- tured alone without cytokines. The P values (AD) were calculated using two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: Cell Culture, Derivative Assay, Concentration Assay

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 4. In vivo function of islets pre-cultured alone, with ANXA1 alone or with a cocktail of MSC secretory factors. (A) Average blood glucose concentrations of STZ diabetic mice trans- planted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (repeated-meas- urements ANOVA with Bonferroni post hoc test, n = 79). (B) Area under the curve (AUC) of STZ diabetic mice transplanted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (one-way ANOVA with Dunn’s post hoc test, n = 79).

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vivo, Cell Culture

Journal: bioRxiv

Article Title: Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

doi: 10.1101/2021.09.23.461563

Figure Lengend Snippet: A) Extracts from untreated (“-“) or pronase-treated (“+”) 1×10 7 strain B31-e2 spirochetes that ectopically produce the indicated surface lipoproteins were separated by SDS-PAGE and transferred to PVDF membranes. The filters were probed with purified C1 complex (top), C1r enzyme (middle) or C1r proenzyme (bottom), and bound probe revealed by anti-C1r antibody, followed by HRP-conjugated anti-mouse antibody. Shown is a representative of 3 experiments. B) Filters prepared identically to panel A were probed with purified C1 complex (top), C1s enzyme (middle) or C1s proenzyme (bottom), and bound probe revealed by anti-C1s antibody, followed by HRP-conjugated anti-mouse antibody. Shown is a representative of 3 experiments. C and D) Biosensors immobilized with GST-ErpB (top) or GST-ErpQ (bottom) were tested by SPR for binding to the indicated concentrations of the enzyme or proenzyme forms of C1r (C) or C1s (D) Injection series were each performed in triplicate. For both panels C) and D), steady-state affinity fits were determined by T200 Biacore Evaluation software and K D values are reported in .

Article Snippet: The next day, membranes were washed in PBS-T and were incubated with α-C1q (Complement Technologies, A200), α-C1r (R&D Systems, MAB1807), or α-C1s (R&D Systems, MAB2060) antibodies, following the manufacturer’s recommended dilutions for western blotting.

Techniques: SDS Page, Purification, Binding Assay, Injection, Software

Journal: bioRxiv

Article Title: Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

doi: 10.1101/2021.09.23.461563

Figure Lengend Snippet: A) Enzymatic cleavage by C1s of the small peptide substrate Z-L-Lys-sBzl was assayed with DTNB (Ellman’s reagent) in the presence of 25 μM BBK32-C (non-inhibitory control) or ErpQ at 25°C for 1hr. Experiments were performed in triplicate. Absorbance was read at 412 nm and signals were normalized to negative control no-substrate wells. B) Top: Proteolytic cleavage of C2 by C1s enzyme produces ∼70kDa C2b and ∼35kDa C2a after 1hr at 37°C. Lanes 1-5: C2b accumulation in the presence (“+”) or absence (“-“) or 25 µM ErpQ, 25 µ M BBK32-C (non-inhibitory control), 6.25 nM C1s, and 685 nM C2. (Note that the amount of C1s loaded is below the level of detection by SDS-PAGE). Lanes 6-13: C2b accumulation in the presence of 6.25 nM C1s, 685 nM C2 and a two-fold dilution series (from 16 to 0.13 μM) of ErpQ. Bottom: The fraction of C2b relative to total input C2 in the same lane determined by densitometry analysis data are normalized to C2 (lane 5) and C1s digested C2 (lane 6). A representative gel is shown. The experiment was performed three times. C) Top: C4, which consists of 3 polypeptide chains, C4α (97 kDa), C4β (77 kDa), C4γ (33 kDa), is cleaved by C1s enzyme for 1hr at 37 °C to produce C4α’ (88 kDa). Lanes 1-5: SDS-PAGE profile in the presence (“+”) or absence (“-“) or 25 µ M ErpQ, 25 µ M BBK32-C (non-inhibitory control), 6.25 nM C1s, and 616 nM C4. Lanes 6-13: SDS-PAGE profile in the presence of 6.25 nM C1s, 616 nM C4 and a two-fold dilution series (from 25 to 0.20 μM) of ErpQ. Bottom: The fraction of C4α’ relative to input C4β in the same lane and normalized to C1s + C4 positive control (lane 6) and negative control C4 (lane 5) was determined by densitometry analysis.

Article Snippet: The next day, membranes were washed in PBS-T and were incubated with α-C1q (Complement Technologies, A200), α-C1r (R&D Systems, MAB1807), or α-C1s (R&D Systems, MAB2060) antibodies, following the manufacturer’s recommended dilutions for western blotting.

Techniques: Control, Negative Control, SDS Page, Positive Control

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis.

doi: 10.1111/j.1538-7836.2006.02033.x

Figure Lengend Snippet: Fig. 4. Complement C3 deposition in joints. Healthy 5–8-week old female TMwt/wt (A) and TMLeD/LeD (B) mice were killed and knee joints were processed for immunohistochemical staining to detect complement factor C3 deposition. These sections are representative of three mice. No C3 was detectable in the joints of TMwt/wt mice (A). C3 was readily observed on the articular surface (small arrows) and in the bone marrow space of TMLeD/LeD mice (large arrows) (B). Higher power views of the articular surface are shown in the right upper corner of each panel.

Article Snippet: Specific immunostaining of histologic sections was achieved by overnight incubation with the following primary antibodies: CD45 (rat, 1:100; BecktonDickinson, Erembodegem, Belgium, no. 553076);Mac3 (rat, 1:300; BecktonDickinson, no. 553322); MPO (rabbit, 1:100, Dako A/S, Glostrup, Denmark, no. A0398); HMGB1 (rabbit; 1:250, Santa Cruz Biotechnology, Boechout, Belgium, no. sc12523); and complement factor C3 (rat, 1:200; Cedarlane,Uden, theNetherlands, no. CL7503AP), which recognizes intact C3, C3b, iC3b and C3d, but not C3a.

Techniques: Immunohistochemical staining, Staining