Journal: PLoS ONE

Article Title: 3D printing and milling a real-time PCR device for infectious disease diagnostics

doi: 10.1371/journal.pone.0179133

Figure Lengend Snippet: (a) The complete device showing locations of Control assembly, Photodiode assembly, Bottom assembly, Case, and Li-Po batteries. (b) Detail of assemblies. The Control assembly contains a faceplate, a control board, a main board, and a MicroView microcontroller. This assembly provides a user interface and houses all electronic controls. The Photodiode assembly contains the photodiode and emission filter. This assembly is used to determine the target DNA concentration present in the sample. The bottom assembly contains a motor, a fan blade, spacers, and a centrifugal fan housing, forming the cooling fan system. It also contains an LED and excitation filter, establishing the light source for illuminating the sample. The cartridge is a removable assembly allowing for easy insertion of the PCR tube containing the sample. It contains the heating block, electrical contacts for connection to the main board, and a vented bottom plate allowing for the escape of warm air.

Article Snippet: The main part of the electronic control system is the SparkFun MicroView.

Techniques: Concentration Assay, Blocking Assay

Journal: PLoS ONE

Article Title: 3D printing and milling a real-time PCR device for infectious disease diagnostics

doi: 10.1371/journal.pone.0179133

Figure Lengend Snippet: (a) The dimension of the qPCR device is 12 × 7 × 6 cm 3 . The MicroView shows the status of amplification cycle and fluorescence reading. (b) The bottom view of the qPCR device showing cartridge bottom and an air inlet for centrifugal fan. (c) The size of the cartridge that holds PCR tube during qPCR is 7.6 × 3.4 × 2.9 cm 3 . (d) Measured fluorescence readings show the shift in the intensity measurements corresponding to the differing concentrations of target virus. The threshold for determining C q is also shown is a dotted line. (e) Measured Cq for three concentrations of target DNA. The mean and standard deviation of C q are 21.74 ± 0.39, 23.66 ± 0.70, and 25.98 ± 1.75, for 2 × 10 7 vp/mL, 2 × 10 6 vp/mL, and 2 × 10 5 vp/mL, respectively.

Article Snippet: The main part of the electronic control system is the SparkFun MicroView.

Techniques: Amplification, Fluorescence, Standard Deviation

Journal: JCI Insight

Article Title: Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles

doi: 10.1172/jci.insight.96910

Figure Lengend Snippet: (A) NHS was incubated with 50 μM hemin, 12.5 μM Hb (1 molecule of Hb contains 4 heme molecules) diluted in TBS. After a 30-minute incubation time at 37°C, the level of released Ba was measured by ELISA. *P < 0.005, Kruskal-Wallis with Dunn’s test for multiple pairwise comparisons. (B) HUVECs were treated with increasing concentrations of hemin or diluted in FCS-free culture medium (M199) for 30 minutes at 37°C. After removing the supernatant, HUVECs were exposed to NHS (33% final concentration diluted in the same medium) for 30 minutes at 37°C. Cells were detached and stained for C3 deposition by flow cytometry. (C–F) HUVECs were treated with increased concentrations of hemin with or without 5 μM of Hx for 30 minutes at 37°C. After removing the supernatant, HUVECs were exposed to NHS (33% final concentration diluted in M199 FCS-free) for 30 minutes at 37°C. Cells were detached and stained for C3 deposition (C and D) or C5b-9 formation (E and F) by flow cytometry. (C and E) Representative flow cytometry histograms. (D and F) Quantitative analyses. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001, 2-way ANOVA with Sidak’s test for multiple comparisons. Values are shown as box plots with median and minimum/maximum points.

Article Snippet: Complement ELISA kits MicroView Ba and sC5b-9 were from Quidel.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Staining, Flow Cytometry

Journal: JCI Insight

Article Title: Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles

doi: 10.1172/jci.insight.96910

Figure Lengend Snippet: (A and B) NHS was incubated with 1,000 MV/μl RBC MVs from HDs (n = 12) or SCD patients (n = 12) diluted in TBS. After 30 minutes at 37°C, the levels of released Ba (A) and sC5b-9 (B) were measured by ELISA. **P < 0.005, Mann-Whitney test. (C) Linear correlation between measured Ba and sC5b-9 levels with RBC MPs from SCD (n = 12) in tested samples. R2 = 0.4811; P = 0.0085. (D and E) NHS was incubated with 1,000 MV/μl MVs from SCD patients (n = 12) diluted in TBS with or without 25 μM Hx. After 30 minutes at 37°C, the levels of released Ba (D) and sC5b-9 (E) were measured by ELISA. *P < 0.05, ***P < 0.001, Wilcoxon test after a Shapiro-Wilk test for normality. (F and G) HUVECs were treated with 1,000 MV/μl RBC MPs from HDs (n = 4) or SCD patient (n = 7) diluted in M199 medium for 30 minutes at 37°C. Without removing the supernatant, HUVECs were exposed to NHS (final concentration at 33% diluted in M199 FCS-free) for 30 minutes at 37°C. Cells were detached and stained for C3 deposition by flow cytometry. (F) Flow cytometry histograms. (G) Quantitative analyses. *P < 0.05, Mann-Whitney test. (H) HUVECs were treated with 1,000 MV/μl RBC MPs from 1 SCD patient with or without 5 μM Hx. As above, NHS was added and cells were stained for C3 deposition by flow cytometry. Values are shown as box plots with median and minimum/maximum points.

Article Snippet: Complement ELISA kits MicroView Ba and sC5b-9 were from Quidel.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Concentration Assay, Staining, Flow Cytometry