Journal: Thrombosis Journal

Article Title: Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm

doi: 10.1186/s12959-023-00549-5

Figure Lengend Snippet: IFE of AMC fluorophore and its correction via calibration or normalization. FVIII-DP was supplemented with 1 IU/mL FVIII to normalize hemophilia plasma, or not, and was subsequently premixed with the indicated concentrations of AMC prior to initiating coagulation with Ca 2+ and substrate. Raw fluorescent data were produced by the CAT microplate reader and software and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) normalized-uncalibrated curves, (G, H) calibrated TG curves (via TS software), (I, J) calibrated TG curves (via OR software), and (L, M) calibrated TG curves (via SH software). Uncalibrated curve data were produced by differentiating the AMC curves observed in (A, B) . Calibrated curves were produced using TS software, our in-house OR software, which uses published algorithms similar to CAT calibration, or SH software, our second in-house app based on CAT algorithm. An asterisk (*) next to the indicated concentrations in panels G & H denotes high AMC concentrations in which commercial TS software did not report TG curves, possibly due to their noisy appearance as suggested by the TG curves reported by OR and SH software apps at these high concentrations. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic sample and normalized hemophilia sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 40–60 min. Assay conditions: 63 µL of FVIII-DP, 1 µL of FVIII (1 IU/mL), 16 µL of AMC at indicated concentrations, 20 µL of PPP trigger, and 20 µL of FluCa.

Article Snippet: The experiment was then initiated by injection of the mixture of provided Fluo-Substrate and Fluo-Buffer (i.e., FluCa reagent mixture) using a Fluoroskan Ascent microplate reader (Stago).

Techniques: Coagulation, Produced, Software, Fluorescence, Concentration Assay

Journal: Thrombosis Journal

Article Title: Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm

doi: 10.1186/s12959-023-00549-5

Figure Lengend Snippet: Substrate consumption and its correction via calibration or normalization. FVIII-DP was supplemented with 1 IU/mL FVIII to normalize hemophilic plasma, or not, and was subsequently premixed with the indicated concentrations of substrate, ZGGR-AMC, prior to initiating coagulation with Ca 2+ and substrate. Raw data was produced by the CAT microplate reader and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) normalized-uncalibrated curves, (G, H) calibrated TG curves (via TS software), (I, J) calibrated TG curves (via OR software), and (L, M) calibrated TG curves (via SH software). Uncalibrated curves data were produced by differentiating the AMC curves observed in (A, B) . Calibrated curves were produced using TS, OR or SH apps all of which employed the same CAT correction algorithm. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic and normalized sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 40 min. Assay conditions: 63 µL of FVIII-DP, 1 µL of FVIII (1 IU/mL), 16 µL of substrate ZGGR-AMC (at indicated concentrations), 20 µL of PPP trigger, and 20 µL of Ca 2+ (calcium chloride buffer)

Article Snippet: The experiment was then initiated by injection of the mixture of provided Fluo-Substrate and Fluo-Buffer (i.e., FluCa reagent mixture) using a Fluoroskan Ascent microplate reader (Stago).

Techniques: Coagulation, Produced, Fluorescence, Software, Concentration Assay

Journal: Thrombosis Journal

Article Title: Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm

doi: 10.1186/s12959-023-00549-5

Figure Lengend Snippet: An attempt to study substrate consumption in plasma samples supplemented with two substrates, ZGGR-AMC and ZGGR-AFC. FVIII-DP was supplemented with 1 IU/mL FVIII to normalize hemophilic plasma, or not, and was subsequently premixed with the indicated concentrations of two substrates, ZGGR-AMC and ZGGR-AFC, such that the ratio of AMC:AFC equaled to a concentration of 800 µM, prior to initiating coagulation with Ca 2+ . Raw data was produced by the Biotek microplate reader and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) Normalized-Uncalibrated curves, (G, H) Calibrated TG curves (via OR software), and (I, J) calibrated TG curves (via SH software). Uncalibrated curve data were produced by differentiating the AMC curves observed in (A, B) . CAT calibrated curves were produced using our in-house OR and SH software apps, which use published algorithms similar to CAT calibration. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic and normalized sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 40–60 min. An artifact resembling TG signal in early minutes in Fig. 5 is only seen with ZGGR-AFC experiments, suggesting that it is caused by either the AFC fluorophore itself (similar to Fig. and Fig. S1 discussed above) or background fluorescence signal of un-cleaved ZGG-AFC substrate. Assay conditions: 78 µL of FVIII-DP, 2 µL of FVIII (1 IU/mL), 20 µL of PPP trigger, and 20 µL of custom FluCa mixtures (substrates ZGGR-AMC and ZGGR-AFC at indicated concentrations with calcium chloride buffer)

Article Snippet: The experiment was then initiated by injection of the mixture of provided Fluo-Substrate and Fluo-Buffer (i.e., FluCa reagent mixture) using a Fluoroskan Ascent microplate reader (Stago).

Techniques: Concentration Assay, Coagulation, Produced, Fluorescence, Software

Journal: Thrombosis Journal

Article Title: Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm

doi: 10.1186/s12959-023-00549-5

Figure Lengend Snippet: Effect of CAT calibration on TG curves in procoagulant plasma samples. Antithrombin deficient plasma (ATIII-DP) was treated with or without heparin and the indicated TF concentration to assess the effect of calibration via different software apps in procoagulant samples. Raw data was produced by the CAT assay microplate reader and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) normalized-Uncalibrated curves, (G, H) calibrated TG curves (via CBER algorithm), and (I, J) calibrated TG curves (via SH software). Uncalibrated curve data were produced by differentiating the AMC curves observed in (A, B) . CAT calibrated curves were produced by our in-house OR and SH software apps. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic and normalized sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 60 min. Assay conditions: ATIII-DP with 0.2 U/mL of normal pooled plasma with or without heparin (0.03 USP/mL), TF (0.12–20 pM), tPA (0.13 µg/mL), thrombomodulin (12.5 nM), PC:PS vesicles (4 µM) and custom FluCa mixture (800 µM ZGGR-AMC and calcium chloride)

Article Snippet: The experiment was then initiated by injection of the mixture of provided Fluo-Substrate and Fluo-Buffer (i.e., FluCa reagent mixture) using a Fluoroskan Ascent microplate reader (Stago).

Techniques: Concentration Assay, Software, Produced, Fluorescence

Journal: PLoS ONE

Article Title: A Putative Non-Canonical Ras-Like GTPase from P . falciparum : Chemical Properties and Characterization of the Protein

doi: 10.1371/journal.pone.0140994

Figure Lengend Snippet: The depicted plot shows the absolute fluorescense.of the formed PfG-Protein-BGTP complex monitored at a wavelength between 480 nm and 510 nm versus increasing protein concentrations with a crude extract of the purified expressed PfG- protein (black square) and a purified enzyme preparation (black circle) after native purificaion. A non-recombinant pET-28a vector (open triangle) and a constitutively expressed Gα s human subunit (open square) were employed as a positive and a negative control. Each point represents the mean value of three different experiments.

Article Snippet: Fluorescense exitation (470 nm) and emission (510 nm) were determined in a fluorescense microplate reader (GoMax-Multi, Promega, Karlsruhe).

Techniques: Purification, Recombinant, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: A Putative Non-Canonical Ras-Like GTPase from P . falciparum : Chemical Properties and Characterization of the Protein

doi: 10.1371/journal.pone.0140994

Figure Lengend Snippet: Part A Absolute Fluorescense of binding of 50 nmol BODIPY FL GTPγS to the plasmodial, PfG-protein (black line). The increase in fluorescence was monitored until saturation over a time interval of 600 s. Part B At the arrow at t = 200 s, 20 μM unlabeled GTPγS was added and the decrease in fluorescence was monitored. The dissociation of BODIPY FL GTPγS was fit with single exponential functions and the half life value was determined i.e. t 1/2 = 9 s suggesting a low affinity for the binding of BODIPY FL GTP. b Monitoring GTP-binding of the P . falciparum G-protein in a time course experiment: Binding assay with different protein concentrations of PfG from Plasmodium green line 62.5 μg, red line 16,5 μg; the reaction was antagonized with unlabeled GTP after 100 s blue line (62,5 μg purified G-protein), purple line (16,5 μg purified-G-protein), yellow line (8,0 μg G-protein).

Article Snippet: Fluorescense exitation (470 nm) and emission (510 nm) were determined in a fluorescense microplate reader (GoMax-Multi, Promega, Karlsruhe).

Techniques: Binding Assay, Fluorescence, Purification

Journal: Nature Communications

Article Title: The mitochondrial ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signalling

doi: 10.1038/ncomms8910

Figure Lengend Snippet: ( a ) Immunoblot analysis of MARCH5 expression levels in MARCH5-depleted Raw264.7 cells. See full blots in . ( b ) Twenty-four hours after infection, PR8-GFP or VSV-GFP virus replication assay by fluorescence microscopy (fluorescence, upper; phase-contrast microscopy, bottom) and virus titration by fluorescence analysis or plaque assay in siControl or siMARCH5 expressing Raw264.7 cells. Error bars, mean±s.e.m. ( n =3). Scale bars, 100 μm. ( c ) Determination of the virus titration using fluorescence analysis or plaque assay in March5 +/+ and March5 +/− BMDM cells infected with PR8-GFP or VSV-GFP virus. Error bars, mean±s.e.m. ( n =3). ( d , e ) Bioassay of IFN-β or IL-6 (ELISA) in supernatants of MARCH5-depleted Raw264.7 ( d ) or March5 +/+ and March5 +/− BMDM cells ( e ) infected or transfected with PR8-GFP, VSV-GFP, poly(I:C) or 5′ppp-dsRNA. After cells were transfected with siControl or siMARCH5 for 24 h, followed by infection with virus or transfection with poly(I:C) for indicated times. Error bars, mean±s.e.m. ( n =3). All data are representative of at least three independent experiments.

Article Snippet: GFP-tagged VSV or H1N1 influenza virus (A/PR8/8/34) replication levels were measured with Fluorescence module of GloMax-Multi Microplate Multimode Reader (Promega).

Techniques: Western Blot, Expressing, Infection, Virus, Fluorescence, Microscopy, Titration, Plaque Assay, Bioassay, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Nature Communications

Article Title: The mitochondrial ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signalling

doi: 10.1038/ncomms8910

Figure Lengend Snippet: ( a ) After Myc-MARCH5 WT or Myc-MARCH5 H43W expression vectors were introduced into MARCH5 −/− HEK293T cells, cells were infected with PR8-GFP or transfected with poly(I:C) for 24 h. Promoter activity of IFN-β, ISRE, IFN-α or IRF3. Graphs represent fold-induction relative to the luciferase activity in control cells. Error bars, mean±s.e.m. ( n =3). ( b ) Immunoblot analysis of endogenous or ectopic expression levels of MARCH5 in MARCH5 +/+ or MARCH5 −/− HEK293T cells. See full blots in . ( c , d ) Twenty-four hours after infection, VSV-GFP replication assay by fluorescence microscopy analysis ( c ). Bioassay of IFN-β or IL-6 production (ELISA) ( d ). Error bars, mean±s.e.m. ( n =3). Scale bars, 100 μm. ( e , f ) IRES, MARCH5 WT -Flag or MARCH5 H43W -Flag stably expressing Raw264.7 cells were stimulated by infection with VSV-GFP for 24 h. Twenty-four hours after infection, VSV-GFP replication assay by fluorescence microscopy analysis (left). Quantification of GFP intensity or virus titration by plaque assay (right; e ). Bioassay of IFN-β or IL-6 production (ELISA) in supernatants of cultured Raw264.7 stable cells ( f ). Error bars, mean±s.e.m. ( n =3). All data are representative of at least three independent experiments. Scale bars, 100 μm.

Article Snippet: GFP-tagged VSV or H1N1 influenza virus (A/PR8/8/34) replication levels were measured with Fluorescence module of GloMax-Multi Microplate Multimode Reader (Promega).

Techniques: Expressing, Infection, Transfection, Activity Assay, Luciferase, Control, Western Blot, Fluorescence, Microscopy, Bioassay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Virus, Titration, Plaque Assay, Cell Culture