Journal: Scientific Reports

Article Title: Novel method for rapid toxicity screening of magnetic nanoparticles

doi: 10.1038/s41598-018-25852-4

Figure Lengend Snippet: ROS intracellular measurements in individual HEK293 cells. Voltammograms before and after penetration of cells with the nanoelectrode. Voltage is applied vs Ag/AgCl. Representative current traces of a nanoelectrode polarized at +800 vs Ag/AgCl inside and outside HEK293 cells. Red and blue arrows indicate, respectively, the moment of penetration and retraction. Top graph - ROS intracellular measurements in cells incubated with Magn NP for 3 h in HBSS, lower graph - ROS intracellular measurements in pure cells after 3 h in HBSS. Grey curves are pure records, black curves are records with 100 averaging.

Article Snippet: To detect ROS in cells after MNPs addition, unfixed cells were washed with HBSS supplemented with 2 mM L-glutamine and 10 mM HEPES (pH 7,4 adjusted with 1 N NaOH), and stained with 2 µM H2DCFDA solution (life technologies) for 30 min at 37 °C in darkness.

Techniques: Incubation

Journal: Scientific Reports

Article Title: Novel method for rapid toxicity screening of magnetic nanoparticles

doi: 10.1038/s41598-018-25852-4

Figure Lengend Snippet: ROS intracellular measurements in individual LNCaP cells. Voltammograms before and after penetration of cells with the nanoelectrode. Voltage is applied vs Ag/AgCl. Representative current traces of a nanoelectrode polarized at +800 vs Ag/AgCl inside and outside LNCaP cells. Red and blue arrows indicated, respectively, the moment of penetration and retraction. Similar traces were obtained from 5 different cells from each sample by the same electrode. Control – ROS intracellular measurements in pure cells after 3 h in HBSS; Magn - ROS intracellular measurements in cells incubated with Magn NP during 3 h, Magn-Plu - ROS intracellular measurements in cells incubated with Magn-Plu NP during 3 h. Grey curves are pure records, black curves are records with 100 averaging.

Article Snippet: To detect ROS in cells after MNPs addition, unfixed cells were washed with HBSS supplemented with 2 mM L-glutamine and 10 mM HEPES (pH 7,4 adjusted with 1 N NaOH), and stained with 2 µM H2DCFDA solution (life technologies) for 30 min at 37 °C in darkness.

Techniques: Incubation

Journal: Experimental Biology and Medicine

Article Title: Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

doi: 10.1177/1535370214558025

Figure Lengend Snippet: Schematic overview of the isolation of PHH and NPC from human liver resections. Liver cells were isolated by a two-step EGTA/collagenase P perfusion technique (1). The cell suspension was centrifuged initially at 50 × g , 5 min, 4℃ (2). PHH (pellet) were separated from the NPC (supernatant). PHH were purified by a Percoll density gradient centrifugation at 1250 × g , 20 min, 4℃ (A and B). The supernatant of PHH isolation (2) was centrifuged first at 300 × g , 5 min, 4℃ (3). KC were found in the supernatant of the first centrifugation step. Cells were treated with a second centrifugation at 650 × g , 7 min, 4℃. Both cell pellets were pooled and re-suspended in HBSS. Afterwards, the cell suspension was transferred to a two-layer (25% / 50%) Percoll density gradient and centrifuged at 1800 × g , 20 min, 4℃ (4). Dead cells on top of the 25% Percoll layer were discarded. NPC were located around the interphase of the 25% and the 50% Percoll layer. KC were separated from the NPC fraction by an adherence separation step (5). LEC and HSC were separated by MACS®. For this purpose, the remaining cells were centrifuged at 300 × g , 5 min, 4℃, and labelled with CD31-conjugated MicroBeads (6). CD31-negative HSC passed the MACS® separation column (7). The column was removed from the magnetic field and the CD31-positive LEC were dissolved from the column (8). (A color version of this figure is available in the online journal.)

Article Snippet: Percoll, Trypan Blue and HBSS were provided by Biochrom (Berlin, Germany).

Techniques: Isolation, Purification, Gradient Centrifugation, Centrifugation, Magnetic Cell Separation

Journal: PLoS ONE

Article Title: Development and Evaluation of a Fluorescent Antibody-Drug Conjugate for Molecular Imaging and Targeted Therapy of Pancreatic Cancer

doi: 10.1371/journal.pone.0157762

Figure Lengend Snippet: In Vivo Tumor Growth Inhibition. Male nude, athymic mice, 4 weeks of age, were subcutaneously inoculated with BxPC-3 cells (2x10 6 ) embedded in 50:50 HBSS:Matrigel. When tumors were palpable (~1 week), mice were tagged and randomly separated into 3 groups. Mice in group 1 (n = 6) received PBS (vehicle). Group 2 (n = 6) received free paclitaxel (6.7uM) in PBS, while group 3 (n = 5) received equimolar amounts of paclitaxel conjugated to antibody (fluorescent ADC, 100ug α-CEA-680-PTX, 6.7uM effective paclitaxel). Each mouse received a total of five doses of the respective treatment, spaced 3 days apart, via retro-orbital injection. Arrows indicate treatment days (days 3, 6, 9, 12, and 15). Tumor size was measured every 3 days using digital calipers, and tumor volume was calculated with the modified ellipsoid formula (v = ½(length × width 2 )). Each data point represents average tumor volume (n = 5–6 mice). and error bars denote standard error.

Article Snippet: Tumor induction After a one week acclimatization period, mice were subcutaneously inoculated with 2x106 BxPC-3 cells embedded in 100uL of 50:50 HBSS:Matrigel (Corning Incorporated, #354262, Corning NY).

Techniques: In Vivo, Inhibition, Mouse Assay, Injection, Modification

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanisms of acute toxicity in NKG2D Chimeric Antigen Receptor T cell treated mice

doi: 10.4049/jimmunol.1600769

Figure Lengend Snippet: Determination of maximum tolerated dose (MTD) for NKG2D CAR T cells (a) overview of approach to determine MTD for single and multi-dose treatment with murine NKG2D CAR T cells. (b) The health score for mice injected with 2 x 10 7 wild type NKG2D T cells (WT) or NKG2D CAR T cells (CH) in non-tumor bearing (upper) and tumor-bearing (lower) mice is shown. Each symbol represents an individual mouse at the time of euthanasia. (c–j) The percent change in initial weight in the days following injection of NKG2D CAR T cells, wild type NKG2D T cells, or HBSS is plotted for 3 to 28 d after T cell injection. Average values for each group are shown (n=10/group). Mice were treated with a single dose of 5 x10 6 T cells (c, d), 10 7 T cells (e, f), or 2 x 10 7 T cells (g, h). Mice were RMA tumor bearing (c, e, g) or non-tumor bearing (d, f, h). (i) RMA tumor-bearing mice (RMA cells i.v. day -5) were injected with 10 7 T cells or HBSS on days 0 and 7. (j) Non-tumor bearing mice were injected with 10 7 T cells or HBSS on days 0, 7 and 14. Data are from two independent experiments (n=10/group). A Student’s t-test was used to compare differences between groups using a two-tailed test assuming unequal variance. An * indicates significant differences compared to WT; * p

Article Snippet: RMA-RG tumor cells (105 cells) in 0.4 mL HBSS (Hyclone) were injected i.v. into C57BL/6 (B6) mice on day 0.

Techniques: Mouse Assay, Injection, Two Tailed Test

Journal: Human Gene Therapy Methods

Article Title: A Rapid Cell Expansion Process for Production of Engineered Autologous CAR-T Cell Therapies

doi: 10.1089/hgtb.2016.120

Figure Lengend Snippet: Optimization of closed transduction process for the manufacture of CD19 CAR-T cells. Lymphocytes were isolated from subject apheresis products using the Sepax II and stimulated as previously described with the following changes. ( A ) After stimulation, PBMCs were seeded at the indicated cell density and transduced in bags as previously described. The transduction efficiency was measured by FACS staining for CD19 CAR expression 4 days post transduction. ( B ) Origen cell culture bags were coated with retronectin at the indicated concentrations. The next day, retronectin was removed and the bags were blocked with 2.5% human serum albumin (HSA) before vector loading. OKT3 activated-PBMCs from three patients (1 × 10 6 cells/mL) were added to separate bags, and transduction efficiency was measured by FACS. ( C ) To evaluate the need for a blocking step following retronectin coating of the bag, bags were coated with 10 μg/mL of retronectin and blocked with either 2.5% HSA or HBSS and then loaded with retroviral vector. No significant difference in transduction was observed by the addition of a blocking step. However, removal of the retroviral vector prior to the T cell transduction did significantly reduce the level of CD19 CAR expression ( p = 0.004, stippled bar). ( D ) In the closed transduction process (control), the bag is coated with retronectin overnight followed by a wash-and-block step prior to vector loading. In an attempt to simplify the process, the block step prior to transduction was removed and the addition of retronectin and vector prior to the addition of cells was combined. In a separate effort, the addition of retronectin, vector, and cells was combined into a single step. All other conditions were the same. All data are representative of at least three independent experiments. Data are presented as mean ± SEM.

Article Snippet: The next day, the retronectin was removed and the bags washed once with 2.5% HEPES in HBSS (Lonza).

Techniques: Transduction, Isolation, FACS, Staining, Expressing, Cell Culture, Plasmid Preparation, Blocking Assay

Journal: Bioscience Reports

Article Title: Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

doi: 10.1042/BSR20140155

Figure Lengend Snippet: Amyloidogenic V122I but not non-amyloidogenic T119M TTR induces the generation of superoxide species and caspase 3/7 activation in human cardiomyocytes ( a ) AC16 cells were treated with V122I TTR, T119M TTR, Antimycin A (positive control) or HBSS (vehicle control) for 24 h. The insults were then removed and the cells loaded with the superoxide-specific reagent DHE as detailed in the experimental section. Red fluorescence produced by DHE oxidation is shown. ( b ) AC16 cells were treated with V122I TTR, T119M TTR or, HBSS for 6 h at 37°C. Cell lysates were prepared as detailed in the experimental section and were added to Ac-DEVD-AFC caspase 3/7 substrate. Fluorescence intensity generated by cleavage of the substrate was measured. Asterisks in both panels indicate statistical significance with P

Article Snippet: When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed in 10 mM phosphate buffer (sodium) pH 7.6/100 mM KCl/1 mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank's balanced salt solution; Mediatech) buffer was used instead.

Techniques: Activation Assay, Positive Control, Fluorescence, Produced, Generated

Journal: Bioscience Reports

Article Title: Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

doi: 10.1042/BSR20140155

Figure Lengend Snippet: Native V122I TTR competes with f-V122I TTR for the same interaction sites in human cardiomyocytes ( a ) AC16 cells were co-incubated with f-V122I TTR (320 nM) and increasing concentrations of amyloidogenic V122I TTR or non-amyloidogenic T119M TTR for 3 h at 4°C. The fluorescence associated with the cells was measured and normalized for total protein content. The data were normalized by the fluorescence from the samples incubated with f-V122I TTR only (100%). Filled triangles, V122I TTR; open circles, T119M TTR. ( b ) AC16 cells were co-incubated with f-V122I (600 nM) and equimolar amounts of unlabelled V122I TTR, T119M TTR, V122I stabilized with a small molecule (V122I-SM) or HBSS. Fluorescence associated with the cells was measured, normalized by total protein content and plotted as percentage with respect to the fluorescence of cells that had been incubated with f-V122I and HBSS only. The data show that similarly to T119M TTR, V122I-SM is unable to efficiently displace cell-associated f-V122I, whereas under the same conditions V122I TTR displaces more than 50% of f-V122I TTR.

Article Snippet: When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed in 10 mM phosphate buffer (sodium) pH 7.6/100 mM KCl/1 mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank's balanced salt solution; Mediatech) buffer was used instead.

Techniques: Incubation, Fluorescence

Journal: Bioscience Reports

Article Title: Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

doi: 10.1042/BSR20140155

Figure Lengend Snippet: Differential interaction of the amyloidogenic V122I TTR and the non-amyloidogenic T119M TTR with the human cardiac cell line AC16 AC16 cells were treated with trypsin (or HBSS as control) to remove membrane-associated proteins. V122I and T119M TTR were then incubated with the cells for 4 h at 4°C or 37°C. Cell lysates were prepared and analysed by SDS—PAGE (15% gel) and Western blot using an anti-TTR and anti-actin antibodies. The lower panel shows densitometry analysis (absorbance) of total cell-associated TTR normalized to actin. Error bars represent S.D. of the biological duplicates. The percentage of TTR/actin ratio in cells pre-treated with trypsin (trypsin +) with respect to control cells (trypsin −) is shown.

Article Snippet: When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed in 10 mM phosphate buffer (sodium) pH 7.6/100 mM KCl/1 mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank's balanced salt solution; Mediatech) buffer was used instead.

Techniques: Incubation, SDS Page, Western Blot

Journal:

Article Title: Analysis of Putative Chlamydia trachomatis Chaperones Scc2 and Scc3 and Their Use in the Identification of Type III Secretion Substrates

doi: 10.1128/JB.187.18.6466-6478.2005

Figure Lengend Snippet: Accessibility of CopB2 to NHS-S-S-biotin in cells permeabilized with staphylococcal alpha-toxin. C. trachomatis -infected (L2) or mock-infected (M) HeLa cultures were treated with HBSS with (+) or without (−) alpha-toxin (α-Toxin),

Article Snippet: HeLa cell monolayers were cultivated in 6-well cluster dishes and mock treated or infected with C. trachomatis L2 for 24 h. Monolayers were washed twice with 37°C HBSS and then treated with 1.0 ml HBSS or HBSS containing 50 μg alpha-toxin (Calbiochem) for 2 h at 37°C.

Techniques: Infection