Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 3. Comparison of HEK cell responses expressing wild-type and mutagenized form of Orco. (a)— Universal Orco agonist, VUAA1, elicits dose-dependent Ca++ i increase in HEK293A cells expressing either CpomOrco (blue) or CpomOrcoQ417H (red). (b)—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation for CpomOrco (blue smooth line) or CpomOrcoQ417H (red smooth line) respectively, providing maximum responses of ~ 6.3 ± 0.1 and ~ 3.9 ± 1.3 ∆F, EC50s of ~ 157.1 ± 3.58 and ~ 261.8 ± 165.6 µM and Hill coefficients of ~ 2.4 ± 0.1 and ~ 2.1 ± 1.9; total number of cells analysed: N = 123 and 94. (c)—Effects of Pear ester on the activity of CpomOrco+OR3 (blue) and CpomOrcoQ417H+OR3 (red) heteromers. (d)—Pear ester concentration dependencies. Data points represent the mean response amplitudes (± SE) of cells from at least three experiments. Data were fit to a Hill equation providing the following parameters: maximum responses of ~ 6.6 ± 0.3 and 3.7 ± 0.1 ∆F; EC50s of ~ 210 ± 14.2 and ~ 733.5 ± 26.9 µM; Hill coefficients of ~ 4.6 ± 2.4 and ~ 4.6 ± 0.4, for CpomOR3/CpomOrco (blue smooth line) or CpomOR3/CpomOrcoQ417H (red smooth line) respectively. Total number of cells analysed: N = 160 and 262. Traces in A and C represent the mean responses of cells from one experiment. Data in B and D were not normalized. Scales in B and D are different.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Comparison, Expressing, Concentration Assay, Activity Assay

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 4. Testing pH sensitivity of HEK cell expressing wild-type and mutagenized form of Orco. (a)— Decrease in fluorescence intensity of the pH sensitive probe, BCECF, possibly reflects acidification of cytoplasm in response to low pH extracellular conditions. (b)—Comparison of VUAA1 concentration dependencies obtained after 30 min incubation at low pHe for CpomOrco or CpomOrcoQ417H. Left panels—VUAA1 activated calcium responses. Right panels—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation with the following parameters: maximum responses of ~ 1.3 ± 0.7 and ~ 1.8 ± 0.2 ∆F; EC50s of ~ 260 ± 187 and ~ 263.3 ± 45.3 µM; Hill coefficients of ~ 2.5 ± 4.3 and ~ 2.2 ± 0.5, for CpomOrco (blue smooth line, n = 84) or CpomOrcoQ417H (red smooth line, n = 63) respectively. Constraints were applied to fit greatly scattered data in B. Traces in B (left panels) represent the mean responses of cells from one experiment. Data in B (right panels) were not normalized. Concentration dependencies obtained in physiologically relevant control conditions were taken from Fig. 3.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Expressing, Fluorescence, Comparison, Concentration Assay, Incubation, Control

Journal: PloS one

Article Title: Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.

doi: 10.1371/journal.pone.0039091

Figure Lengend Snippet: Figure 1. WAS gene expression is restricted to hematopoietic cells and hemogenic progenitors. (A) Analysis of WAS gene expression in different cell lines. mRNA was obtained from hematopoietic cell lines (AlloT and Raji), hESCs-derived myeloid CFUs (And-1 CFUs), undifferentiated hESCs (AND-1 and SHEF-2), a human fibroblastic cell line (293T) and human endothelial cells (HUVEC) and analyzed by RT-PCR for WAS expression (see M&M for details). (B) Time-course analysis of WAS gene expression during hematopoietic differentiation of AND-1 and H9 hESCs (see M&M and Figure S1 for details). mRNA was extracted at different days during differentiation as indicated and WAS expression analyzed by RT-PCR. (C) Analysis of WAS gene expression in hemogenic progenitors and hematopoietic cells. CD452CD31+ (containing hemogenic progenitors) and CD45+CD31+

Article Snippet: Cell Lines and Culture Media 293T cells (CRL11268; American Type Culture Collection; Rockville, MD) were grown in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Edinburgh, Scotland) with GlutaMAXTM and supplemented with 10% Fetal Bovine Serum (FBS) (PAA Laboratories GmbH, Austria).

Techniques: Gene Expression, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Gene therapy

Article Title: Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection

doi: 10.1038/gt.2011.102

Figure Lengend Snippet: Production of gRV on 293T versus 293F cells. (a) Titer of an LV vector (pRRL.PPT.SF.GFP.pre*), LTR-driven gRV vector (MIEG3) and an SIN gRV vector (SIN11.SF.eGFP.pre*) pseudotyped with an ecotropic envelope derived from 293T and 293F cells transfected with Lipofectamine under adherent conditions (mean±s.d., n=2, P=0.05). (b) Titer of gRV vector (MIEG3) pseudotyped with the RD114 envelope derived from 293T and 293F cells after transfection with Ca-phosphate, Lipofectamine 2000 or Fecturin under adherent conditions (mean±s.d., n=2, P<0.05).

Article Snippet: 51 Cell culture and media HEK293 cell-derived 293T cells (ATCC, Manassas, VA, USA) were thawed and expanded in DMEM supplemented with 10% FBS.

Techniques: Plasmid Preparation, Derivative Assay, Transfection

Journal: Gene therapy

Article Title: Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection

doi: 10.1038/gt.2011.102

Figure Lengend Snippet: The 293T cells cultured on Fibra-Cel. Microscopic image of 293T cells on a Fibra-Cel disk. Disks had been rinsed with phosphate-buffered saline, treated with trypsin and rinsed in DMEM to remove the bulk of the cells. Cells were fixed with 70% isopropanol and stained with 0.4% Trypan blue (×400).

Article Snippet: 51 Cell culture and media HEK293 cell-derived 293T cells (ATCC, Manassas, VA, USA) were thawed and expanded in DMEM supplemented with 10% FBS.

Techniques: Cell Culture, Saline, Staining

Journal: Gene therapy

Article Title: Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection

doi: 10.1038/gt.2011.102

Figure Lengend Snippet: Expansion and transfection of 293T cells on Fibra-Cel. (a) Comparison of infectious titer of SRS11.SF.DsRed2.pre* (GALV pseudotyped) from transfected 293T cells that had been expanded in T75 flasks or on Fibra-Cel. (b) Decline in the level of glucose over time after seeding 2.5×108 293T cells in a bioreactor containing Fibra-Cel in 1 l DMEM 10564 with 10% FBS. Glucose levels from days 0 to 7 (inset) and during last 24 h (graph). (c) Infectious titer of SRS11.SF.GFP.pre*SE (GALV-pseudotyped) vector (harvests 1 and 2) generated by 293T cells transfected on Fibra-Cel in a bioreactor at a 1 l scale (average±s.d., triplicate titration). Transfection mixture contained 1 mg SRS11.SF.GFP.pre*SE, 0.9 mg gag/pol and 0.4 mg GALV plasmid (2.3 μg ml−1 total plasmid concentration). (d) Relative amount of infectious virus collected from transfected 293T cells in tissue culture flasks (n=34) or on Fibra-Cel (n=9) using five alternating 10- and 14-h harvests (average±s.e.m.). Harvests 1–5 were collected at 43, 53, 67, 77 and 91 h post-transfection. Vectors included: SRS11.SF.GFP.pre* (GALV pseudotyped; n=4), RSF91.DsRed2.pre* (Eco pseudotyped; n=2), SRS11.SF.DsRed2.pre* (Eco and GALV pseudotyped, n=30) and SRS11.EFS.IL2RG.pre* (Eco, GALV, RD114 pseudotyped, n=7).

Article Snippet: 51 Cell culture and media HEK293 cell-derived 293T cells (ATCC, Manassas, VA, USA) were thawed and expanded in DMEM supplemented with 10% FBS.

Techniques: Transfection, Comparison, Plasmid Preparation, Generated, Titration, Concentration Assay, Virus

Journal: Gene therapy

Article Title: Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection

doi: 10.1038/gt.2011.102

Figure Lengend Snippet: Cell culture history, timing and temperature. (a) Relative infectious titer of gRV vector produced by 293T cells that had been seeded at different cell densities 4 days before transfection. At the day of transfection, cells were harvested and 2×108 cells from each group were transfected on Fibra-Cel. Titers are normalized relative to the 2.5×104 cells per cm2 group (four experiments; average±s.d., P<0.05). Vectors included SRS11.EFS.IL2RG.pre* (GALV pseudotyped) (n=1); MIEG3 (Eco pseudotyped) (n=2); and SIN11.SF.eGFP.pre* (GALV pseudotyped) (n=1). (b) Relative amount of infectious gRV vector produced in tissue culture flasks after media change at different times post-transfection. Results from two independent experiments were combined and expressed as a percentage relative to a common time point (31 h). Vectors used: SRS11.EFS.EGFP.pre* (GALV pseudotyped) and SRS11.EFS.IL2RG.pre* (GALV pseudotyped). (c) Total infectious units collected from 293T cells transfected on Fibra-Cel with the SRS11.SF.GFP.pre* (GALV-pseudotyped) vector at two temperatures (average±s.d., n=2 experiments, P<0.05). (d) Total infectious units from 293T cells transfected on Fibra-Cel with the SRS11.SF.DsRed2.pre* (Eco-pseudotyped) vector in the presence of increasing concentrations of total plasmid DNA (average±s.d., n=3 experiments each for 6.9 and 9.2 μg ml−1 groups).

Article Snippet: 51 Cell culture and media HEK293 cell-derived 293T cells (ATCC, Manassas, VA, USA) were thawed and expanded in DMEM supplemented with 10% FBS.

Techniques: Cell Culture, Plasmid Preparation, Produced, Transfection

Journal:

Article Title:

doi:

Figure Lengend Snippet: LPS peptide mimics were dissolved in endotoxin free water and added to 96-well plates at three concentrations; 1 µg/ml (black bar), 5 µg/ml (gray bar), and 10 µg/ml (white bar). HEK-BLUE™-4 were then added to each well at a concentration of 5×10 4 cells per well in complete media. The supernatant was then harvested after 24 h and assayed for alkaline phosphatase secretion indicating activation of NF-κB. NF-κB activation for peptides RS01-RS07 is represented in and peptides RS08-RS12 in . LPS was used as a positive control in assaying all peptides for NF-κB activation. Absorbance was taken at 630 nm. Each experiment was repeated three times with similar results observed and the standard deviation shown above each sample represents three replicates in one experiment.

Article Snippet: HEK-BLUE™-4 cells were purchased from Invivogen and were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), penicillin 10,000 IU/ml, streptomycin 10,000 µg/ml (Mediatech), Normocin™ 100 µg/ml (Invivogen), 1× HEK-BLUE™ selection antibiotics mixture (2 ml/500 ml complete media) (Invivogen), and 2 mM L-glutamine (Mediatech).

Techniques: Concentration Assay, Activation Assay, Positive Control, Standard Deviation

Journal:

Article Title:

doi:

Figure Lengend Snippet: Nuclear translocation studies of NF-κB were performed by Western blot analysis on both RAW264.7 , HEK-BLUE™-4 , and HEK293 cells. Cells were stimulated with peptide, either RS01or RS09, at a concentration of 5 µg/ml per 5×10 4 cells at various time points (15, 30, 60, and 120 min). Nuclear protein fractions were assayed for NF-κB while cytoplasmic proteins fractions were assayed for both NF-κB and IκB-α. HDAC was used as a nuclear protein loading control and actin was used as a cytoplasmic protein loading control. For HEK293 cells, no differences in the levels of cytoplasmic and nuclear NF-kB were observed suggesting that LPS, RS01, and RS09 do not activate the non-TLR-4 expressing HEK293 cells .

Article Snippet: HEK-BLUE™-4 cells were purchased from Invivogen and were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), penicillin 10,000 IU/ml, streptomycin 10,000 µg/ml (Mediatech), Normocin™ 100 µg/ml (Invivogen), 1× HEK-BLUE™ selection antibiotics mixture (2 ml/500 ml complete media) (Invivogen), and 2 mM L-glutamine (Mediatech).

Techniques: Translocation Assay, Western Blot, Concentration Assay, Expressing