BCG Matrix Analysis Case Solution

BCG Matrix Analysis, Genomic Study, and Single-Cell Sequencing {#sec2.6} —————————————————————- Following three to five rounds of single-cell isolation, we proceeded to perform cell-by-cell nucleofection to label all the cells for next-generation genomic studies. Cells were sorted into 75 cm^2^ tissue culture flasks using the JE-4.

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5 nozzle system (Biovista, Herrsching, Germany). Neutrophils were separated from other cell types using the BD Facssort system and the BD Aria III system both from BD Biosciences (Palo Alto, CA). After loading sorted cells (flowrate ∼6 mL/min) into prelabeled 10 cm sterile culture dishes, we performed 15 to 20 rounds of centrifugation at 300*g*.

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After the final centrifugation step, we collected residual cell pellets for DNA extraction. We removed excess culture medium from the cell pellets by aspirating them using 15-ml syringes fitted with 25G needle and 8 G syringe, followed by spin down at 2000*g* for 2 min. We lysed \~50 µL of suspension by adding 800 µL of 50 mM Tris, pH 8.

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0 containing 50% (w/v) CTAB solution, followed by addition of 900 µL of 100 mM NaCl (final addition of NaCl), 700 µL of 80% (v/v) ethanol, 200 µL of 0.2 M EDTA (final addition of EDTA), and finally 400 ml of 10 mM Tris, pH 8.0 in a heating block at 65°C for 10 min following the manufacturer\’s instructions for a single-cell lysis.

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To increase cell lysis efficiency, we skipped the initial step, which is the addition of the EDTA, and performed the following steps in the same tubes and also concurrently. Lastly, we extracted nucleic acids from the cell lysates by adding 180 µL of ISOLATE II DNA kits (Bioline, London, UK). To perform the cacion-pipemization step, we first added 50 µL of 40 µM random decamer d(T~12–18~T~12–18~) (IDT, Coralville, IA) to a given tube.

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The ISOLATE II DNA kit concentrations of CTAB and ISOLATE II were found to be optimal in terms of percentage of remaining organic components during extraction. Following 10 cycles of pipetting up and down to resuspend remaining particles, we pipetted 400 ul of the nucleic acid elution down the columns. Following a final wash step, we reverse-transcribed the RNAse H treatment using random hexamers and transcriptase.

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We performed the process twice in batches, and each cycle involves 10 µL amplification in parallel using the Thermostar Gradient Master mix (5×) buffer (Agilent, Santa Clara, CA), and 50 ng of cDNA template. Following the same amplification round with a randomized set of primers (with and without transcriptase), we reverse-transcribed the cBCG Matrix Analysis {#s2b} ——————— We first examined the *BD-1* mRNA levels resulting from H~2~S treatment. Treatment of K562 cells with H~2~S (300 µM) resulted in a 5.

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3 fold increase in BD-1 mRNA level 1 hour later with respect to the untreated control ([fig. 2](#pone-0081418-g002){ref-type=”fig”}). ![Effects of H~2~S on BD-1 mRNA level in K562 cells.

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\ K562 cells were treated with H~2~S at the indicated concentrations, total mRNA was isolated after 0, 1, 2, 4 and 8 hours of treatment and levels of BD-1 mRNA were evaluated by real-time RT-PCR using BD-1 specific primers and sequences. The results presented are from densitometry analyses performed on three independent reactions, averaged and expressed as mean ±SD for three independent experiments in triplicates. \*\*\*p\<0.

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001. (n = 3).](pone.

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0081418.g002){#pone-0081418-g002} We then treated two HL-60 cell lines and two primary CD34+ cells with 10 µM of H~2~S for 2 hours and analyzed mRNA levels of BD-1 in a time course of time ([fig. 3A,B](#pone-0081418-g003){ref-type=”fig”}).

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As we anticipated that H~2~S treatment, under the above used concentration, did not reach cytotoxic levels in HL-60, HU-77 and K562 ([fig. S2](#pone.0081418.

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s002){ref-type=”supplementary-material”} A–C), we analyzed BD-1 mRNA levels in 2 hours of H~2~S treatment. In primary CD34+ cells, we found an increase of BD-1 mRNA level in 2 hours of H~2~S treatment, and achieved 2- to 3-fold increase in 2 and 4 hours of treatment. A basal increase of BD-1 mRNA was observed 1 hour after exposure to H~2~S.

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BD-1 mRNA level remained unchanged in HL-60 for at least 8 hours treatment find this in HU-77 and K562 for 24 hours. ![Effects of H~2~S on BD-1 mRNA level in two HL-60 cell lines and two primary CD34+ cells.\ (A) Experiments were performed as in [Figure 2A](#pone-0081418-g002){ref-type=”fig”}, gene expression for BD-1 was evaluated in two HL-60 cell lines treated or not with 10 µM of H~2~S for 2 hours and 2 and 4 hours respectively (B) Experiments were performed as in [fig.

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2A](#pone-0081418-g002){ref-type=”fig”}, and treated as depicted in (A), K562 was treated with 10 µM of H~2~S for 4 hours and in one additional experiment K562 was treated with 10 µM of NaHS (100 µM) for 8 hours. Results presented are from densitometry analyses performed on three independent reactionsBCG Matrix Analysis {#sec2-sensors-20-00292} ——————- The measurements of the Bacterial Contamination Test were obtained in four phases. [Table 1](#sensors-20-00292-t001){ref-type=”table”} summarizes all of the parameters measured in the different measurement phases.

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Phase 1 (first level of the test) was performed with a low dose of indicator bacteria (50% of the liquid volume being a bacterial culture) in the testing chamber. The medium of bacteria from the test chamber is then mixed with the medium of the Bacteriological Reference Material (B. R.

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M.) VITI-500, both without oxygen. The reference bacteriological media is identical to the test media.

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The next phase is the test with the medium of bacteria being mixed with the medium of the B. R. M.

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VITI-500 and with exposure to moderate (60%) and high concentrations of oxygen (100%). Finally the test is continued with a lower oxygen concentration (50%) and a higher bacterial concentration (99%). The measured phase 1 parameters are taken from the following expressions:$${OXY:}Oxygen = \frac{C_{O_{2}}}{C_{C}}$$ where:$OXY$: the concentration of oxygen;$C_{C}$: the concentration of culture of target bacteria (C$_{C}$ = 1.

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33 x 10^4^),$C_{O_{2}}$: the concentration of oxygen in the testing chamber (C$_{O_{2}}$ = 9500 ppm). Secondly, [Table 2](#sensors-20-00292-t002){ref-type=”table”} presents the parameters measured in three, five, and seven times phases of the test. By running the test four times in every single phase (each phase has a lower dose), a 15× the initial dose increases the frequency of sampling by a factor of about 11.

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The measured parameters for two times increase (15-times) are presented in [Table 3](#sensors-20-00292-t003){ref-type=”table”}. 2.4.

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Data Interpretation and Results {#sec2dot4-sensors-20-00292} ———————————– The purpose of the tests was the measure and characterize the susceptibility of the flocculant (Microsep-5) to its interaction with the B. R. M.

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VITI-500 medium which does not contain O$\left( {{NO}_{3}^{-},{NH}_{4}} \right)$. The structure and properties ofMicrosep-5 particles were examined with scanning electron microscopy (SEM) and optical microscopy observation using transmission and wide-field microscopy. These results were used to draw conclusions regarding the flocculant’s physical structure.

### 2.4.1.

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SEM Microscopy in CNT-A–Polyurethane Membranes {#sec2dot4dot1-sensors-20-00292} The flocculant Microsep-5 particles suspended in air inside a CNT-A Membrane showed characteristics typical for fine (diameter below 1 µm) microspheres. Some of the particles were 3-$\mu$m in size, others 5-$\mu$m and with a few larger particles (between 10–23$\mu$m). These microspheres showed a spherical shape and a smooth surface with regular topography.

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A closer inspection of [Figure 7](#sensors-20-00292-f007){ref-type=”fig”}, indicates that Microsep-5 aggregates are composed of spherical particles, i.e., flocs.

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This fact is reflected in the typical particle diameter, which is close to the size of droplets in water. ### 2.4.

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2. SEM Microscopy in Glass Filter Media with Plastic Coating {#sec2dot4dot2-sensors-20-00292} Small unilamellar particles made of Microsep-5 were observed in the surface of the plastic coating in [Figure 8](#sensors-20-00292

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