Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Poly(ADP-Ribose) Glycohydrolase (PARG) Silencing Suppresses Benzo(a)pyrene Induced Cell Transformation

<div><p>Benzo(a)pyrene (BaP) is a ubiquitously distributed environmental pollutant and known carcinogen, which can induce malignant transformation in rodent and human cells. Poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme that catalyzes the degradation of poly(ADP-ribose) (PAR), has been known to play an important role in regulating DNA damage repair and maintaining genomic stability. Although PARG has been shown to be a downstream effector of BaP, the role of PARG in BaP induced carcinogenesis remains unclear. In this study, we used the PARG-deficient human bronchial epithelial cell line (shPARG) as a model to examine how PARG contributed to the carcinogenesis induced by chronic BaP exposure under various concentrations (0, 10, 20 and 40 μM). Our results showed that PARG silencing dramatically reduced DNA damages, chromosome abnormalities, and micronuclei formations in the PARG-deficient human bronchial epithelial cells compared to the control cells (16HBE cells). Meanwhile, the wound healing assay showed that PARG silencing significantly inhibited BaP-induced cell migration. Furthermore, silencing of PARG significantly reduced the volume and weight of tumors in Balb/c nude mice injected with BaP induced transformed human bronchial epithelial cells. This was the first study that reported evidences to support an oncogenic role of PARG in BaP induced carcinogenesis, which provided a new perspective for our understanding in BaP exposure induced cancer.</p></div

PARG silencing inhibited BaP-induced cell motility.

<p>Wound-healing assay of cells treated with different concentrations BaP for 1, 9 or 15 weeks. Representative images of cell migration were shown in <b>A</b>. Migration of the wound edge was measured at ten randomly chosen points in the photograph. The results were quantified as shown in <b>B</b>. Cell migration distances after 24 hrs ten randomly chosen points were compared with the distances at 0 h. Data were shown as mean±S.D. of triplicate measurements. <i>P</i> values were determined by Student’s <i>t</i> test. (* <i>p</i><0.05 and ** <i>p</i><0.01, BaP-treated groups compared with untreated groups; ^# <i>p</i><0.05, a significant difference between two different cells under the same condition).</p

PARG silencing down-regulated cell colony growth induced by BaP exposure.

<p><b>A</b>, Protein expression of PARG in shPARG and 16HBE cell which cultured at the 15th week was determined by immunoblotting. <b>B</b>, Protein expression of PAR in shPARG cell which cultured at the 15th week treated with 40 μM BaP for 24 hrs. Detection of total GAPDH was used to verify equal protein loading. <b>C</b> and <b>D</b>, the two different cell lines were assessed for transformation ability in soft agar assay after treatment with 40 μM BaP for 1, 9 and 15 weeks. Cells (3×10³) were seeded in 1 ml of 0.3% Basal Medium Eagle (BME) agar containing 10% calf serum (CS). The cultures were maintained at 37°C in 5% CO₂ atmosphere for 3 weeks and then colonies were counted using Image-Pro PLUS (v.6) software and representative images were shown <b>(C)</b>. The average colony number was calculated from three separate experiments and data were shown as mean±S.D. <b>(D)</b>. Significant differences were evaluated using the Student’s <i>t</i> test, and data were presented as mean±S.D. of triplicate experiments. The respective asterisks indicated a significant change in BaP-treated cells compared with the untreated controls (*, <i>p</i>< 0.05; **, <i>p</i>< 0.01) and # indicated a significant change between two different cells under the same condition (<i>p</i><0.05).</p

PARG silencing protected against BaP-induced DNA damage.

<p>DNA damage of cells was detected by comet assay after treatment with different concentrations of BaP for 1, 9 or 15weeks. Representative DNA comet images were shown <b>(A)</b> and the average tail moment was calculated from three separate experiments. Data were shown as mean±S.D. (<b>B</b> and <b>C</b>). Statistical differences were evaluated using the Student’s <i>t</i> test. The asterisks (* and **) indicated a significant difference (*<i>p</i>< 0.05 and **<i>p</i><0.01, respectively) between the BaP-treated groups compared with untreated groups. ^# indicated a significant difference (<i>p</i><0.05) between two different cells under the same condition.</p

PARG silencing suppressed tumor formation of BaP-induced transformation cells in nude mice.

<p>Cells transformed by BaP exposure had tumorigenic activity in nude mice. The 16HBE cells and shPARG cells induced by 40 μM BaP for 15 weeks and untreated control cells were injected subcutaneously in nude mice. The nude mice were photographed after cells injection for 4 weeks and the tumors were excised <b>(A)</b>. Average tumor volume <b>(B)</b> and weight <b>(C)</b> were shown. Data were represented as mean±S.D. and significant differences were evaluated using the Student’s <i>t</i> test (* <i>p</i> < 0.05, BaP-treated groups compared with untreated groups; ^# <i>p</i><0.05, a significant difference between two different cells under the same condition). <b>D</b>, H&E stained of the tumor tissue and imaged by the Image-Pro PLUS (v.6) computer software program (400×). <b>E</b>, Protein expression of PARG (green) in tumors from nude mice was examined by immunofluorescence staining and DNA was stained with DAPI (blue). Scale bar:20 μm.</p

PARG silencing decreased BaP-induced micronuclei formation.

<p>Representative photo micrographs were shown as following: <b>A</b>, Normal mononucleated (m), binucleated (b), andtrinucleated (t) cells without micronuclei (100×). <b>B</b>, Binucleated cell without micronuclei (1000×). <b>C</b>, Multiple micronuclei (arrows) in binucleated cells (1000×). <b>D</b>, The average of micronuclei in binucleated cells was calculated from three separate experiments and data were shown as mean±S.D.. Statistical differences were evaluated using the Student’s <i>t</i> test and asterisks (* and **) indicated a significant difference (* <i>p</i><0.05 and ** <i>p</i><0.01, respectively) between the BaP-treated groups compared with untreated groups. ^# indicated a significant difference (<i>p</i><0.05) between two different cells under the same condition.</p

PARG silencing decreased BaP-induced chromosomal aberrations.

<p>Representative metaphase spread of cells stained with Wright-Giemsa stain. <b>A</b>, Normal chromosome; <b>B</b>, Dicentrics; <b>C</b>, Ring chromosome; <b>D</b>, Breaking of chromatid; <b>E</b>, Fragmentation; <b>F</b>, Polyploidy in chromosomes. <b>G</b>, The average of chromosomal aberrations was calculated from three separate experiments and data were shown as mean±S.D.. Statistical differences were evaluated using the Student’s <i>t</i> test and the asterisks (* and **) indicated a significant difference (* <i>p</i><0.05 and ** <i>p</i><0.01, respectively) between the BaP-treated groups compared with untreated groups. ^# indicated a significant difference (<i>p</i><0.05) between two different cells under the same condition.</p