pbs  (Corning Life Sciences)

Journal: Molecular Biology of the Cell

Article Title: Chromosomal passenger complex hydrodynamics suggests chaperoning of the inactive state by nucleoplasmin/nucleophosmin

doi: 10.1091/mbc.E16-12-0860

Figure Lengend Snippet: Tagging of CDCA8 in HeLa cells with GFP-3xFLAG. (A) HeLa cells were arrested in mitosis by overnight treatment with STLC and collected via mitotic shake-off, followed by washing with PBS. The indicated drugs were added to the final wash of the cells, after which the cells were lysed with freeze–thaw cycles. Cell lysates were then immunoblotted for pT232-AURK (recognizing pAURKA, pAURKB, and pAURKC) and α-tubulin. The p value between the barasertib- and okadaic acid–treated cells is 0.069; n = 2. (B) Lysates of the parent and tagged cell line were prepared as in A. Magnetic beads preloaded with an antibody against FLAG were used to immunoprecipitate (IP) tagged proteins from the lysates, and the resulting proteins were blotted for FLAG after separation via SDS–PAGE gel electrophoresis; n = 2. (C) Lysates of the tagged cell line were prepared as in A, and IPs were done with beads loaded with antibodies against random IgG or FLAG, followed by blotting for FLAG as in A; n = 2. (D) HeLa cells expressing GFP-tagged CPC were fixed with 4% paraformaldehyde, stained with an antibody against tubulin, and imaged with a spinning-disk confocal microscope at 60× magnification. Scale bars, 10 μm. Cells were imaged on three separate occasions.

Article Snippet: The mitotic cells were washed three times with cold PBS (Corning) supplemented with STLC.

Techniques: Magnetic Beads, SDS Page, Nucleic Acid Electrophoresis, Expressing, Staining, Microscopy

Journal: bioRxiv

Article Title: cDC1 Coordinate Innate and Adaptive Responses in the Omentum required for T cell Priming and Memory

doi: 10.1101/2020.07.21.214809

Figure Lengend Snippet: An IL-12 independent role for cDC1s in the CD8 + T cell response to CPS (A-I) WT and Batf3 -/- mice were immunized with 10 5 CPS parasites followed by i.p. administration of either PBS or 200 ng rIL-12 at 0, 24, and 48 hpi. T cell responses in the peritoneum were assessed via flow cytometry at 11 dpi. (A) Representative plots of AS15:I-A b+ CD4 + T cells. (B-C) The fraction and number of AS15:I-A b+ CD4 + T cells. (D) Representative plots of tgd057:K b+ CD8 + T cells. (E-F) The fraction and number of tgd057:K b+ CD8 + T cells. (G)Representative plots of the T eff and Tmem phenotypes of tgd057:K b+ CD8 + T cells. (H-I) The fraction and number of Tmem or T eff phenotypes of tgd057:K b+ CD8 + T cells. Data (n = 3-5) represent 1 experiment out of 2 independent experiments. All representative plots indicate mean ± SD. All statistical comparisons were unpaired Student’s t test. ns, not significant; *p

Article Snippet: For IL-12p70 add back experiments, C57BL/6 and Batf3 -/- mice were treated with an intraperitoneal (i.p.) injection of either PBS (Corning: 21-0311-CM) or 200 ng of recombinant murine IL-12p70 (Peprotech: 210-12) immediately after immunization and once per day for the next two days after immunization.

Techniques: Mouse Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Screening for C3 Deficiency in Newborns Using Microarrays

doi: 10.1371/journal.pone.0005321

Figure Lengend Snippet: Visualization of the fluorescence intensitites of the microarrays. The upper part shows a subset of the 269 control DBSS. The Swedish C3 deficient and C3 positive serum standards contains 23 serial dilutions each (high to low dilution, 1∶5 to 1∶100 000). The two lowest dilutions of the standard curve are separated by a blank (PBS). The dotted rectangle depicts the C3 deficient sample.

Article Snippet: For determination of C3 levels by microarrays, the serum samples were diluted 1∶100 in phosphate-buffered saline (PBS) with 0.5% Tween20 and printed onto Epoxide slides (Corning, the Netherlands) using a non-contact printing robot (Nano-plotter 2.0, Gesim, Germany), which deposits approximately 0.4 nl/drop.

Techniques: Fluorescence

Journal: Nucleic Acids Research

Article Title: An optimized, broadly applicable piggyBac transposon induction system

doi: 10.1093/nar/gkw1290

Figure Lengend Snippet: Schematic illustration of the ERT2 and destabilized domain (DD) based PiggyBac (PB) transposon induction systems. ( A ) For the ERT2-based PB transposon induction system, the PBase-ERT2 fusion is constitutively expressed but sequestered outside of the nucleus by HSP90, which binds the ERT2 domain. In the presence of ER antagonist 4-OHT, HSP90 dissociates and the PBase-ERT2 fusion rapidly relocates to the nucleus where it directs transposition. ( B ) For the DD-based PB transposon induction system, a DD (either FKBP or DHFR) was fused to the PBase. The DD confers the instability to the fused protein such that the PBase fusion protein was constitutively degraded. However, binding of a small molecule ligand (Shld1 for FKBP; TMP for DHFR) to the DD prevents PBase from degradation and stabilizes it.

Article Snippet: To select cells in which the PB transposon transposed, cells were trypsinized 2 days after transfection and cultured on 10 cm dish (Corning) with 10 ml fresh media containing puromycin (1 μg/ml).

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: An optimized, broadly applicable piggyBac transposon induction system

doi: 10.1093/nar/gkw1290

Figure Lengend Snippet: Optimization of the induced transposition activity and fold induction for FKBP-based PB transposon induction system. ( A ) Induced transposition activity of different PBase fusion proteins relative to wild-type PBase across four cell lines. Experiments were done in triplicates. The induced transposition activity of a PBase fusion protein was calculated as the normalized number of colonies from the PBase fusion in the presence of corresponding chemical inducer divided by that from ‘wild type’ unfused PBase. ( B ) Fold induction of different PBase fusion proteins across four cell lines. The induction fold was calculated as the normalized number of colonies from chemical inducer treated samples divided by that from untreated samples. ( C ) Comparison of the induced transposition activity with the fold induction for different PBase fusion proteins across four cell lines.

Article Snippet: To select cells in which the PB transposon transposed, cells were trypsinized 2 days after transfection and cultured on 10 cm dish (Corning) with 10 ml fresh media containing puromycin (1 μg/ml).

Techniques: Activity Assay

Journal: Nucleic Acids Research

Article Title: An optimized, broadly applicable piggyBac transposon induction system

doi: 10.1093/nar/gkw1290

Figure Lengend Snippet: The performance of the cumate-regulated FKBP-based PB transposon induction system. Cumate was included in culture media at concentrations ranging from 70 μg/ml (full induction of the cumate promotor) to 0 μg/ml (no induction). For each cumate concentration, Shld1 (yellow) or vehicle (70% ethanol, blue) was added to the medium to activate the PB transposon induction system. The normalized number of puromycin-resistant colonies and the fold-activation are plotted at different cumate/Shld-1 concentrations.

Article Snippet: To select cells in which the PB transposon transposed, cells were trypsinized 2 days after transfection and cultured on 10 cm dish (Corning) with 10 ml fresh media containing puromycin (1 μg/ml).

Techniques: Concentration Assay, Activation Assay

Journal: Nucleic Acids Research

Article Title: An optimized, broadly applicable piggyBac transposon induction system

doi: 10.1093/nar/gkw1290

Figure Lengend Snippet: The performance of different helper constructs in DD and ERT2 based PB transposon induction systems. ( A ) Typical images of colony forming and staining assays to evaluate the transposition efficiency. The scale bar in the enlarged image equals 0.1 mM. The construct used is FKBP-PBase. (a) Cells transfected with donor and helper backbone plasmids (yeast shuttle vector pRS314) were used to estimate the background or random insertions. (b and c) Cells transfected with FKBP-PBase and donor plasmids either in the absence or presence of Shld1 were used to evaluate the transposition efficiency. (d) Cells transfected with unfused PBase and donor plasmid were used to estimate the maximum transposition efficiency. ( B ) Induced PBase activity of different PBase fusions relative to wild-type PBase across four cell lines. The number of puromycin-resistant colonies formed from the cells transfected with both donor and helper plasmids was normalized to that with donor and helper backbone plasmids prior to any further calculations (background subtraction). The induced transposition activity of an inducible domain (i.e. FKBP, DHFR or ERT2) fused PBase was calculated as the normalized number of colonies from the PBase fusion divided by that from ‘wild type’ unfused PBase. Experiments were done in triplicate. ( C ) Non-induced PBase activity of different PBase fusions across four cell lines. Experimental conditions were the same as in B except that no drug was added for the non-induced samples. ( D ) Fold induction of different PBase fusions across four cell lines. The induction fold was calculated as the normalized number of colonies from induced samples in B divided by that from untreated samples in C.

Article Snippet: To select cells in which the PB transposon transposed, cells were trypsinized 2 days after transfection and cultured on 10 cm dish (Corning) with 10 ml fresh media containing puromycin (1 μg/ml).

Techniques: Construct, Staining, Transfection, Plasmid Preparation, Activity Assay

Journal: Nucleic Acids Research

Article Title: An optimized, broadly applicable piggyBac transposon induction system

doi: 10.1093/nar/gkw1290

Figure Lengend Snippet: Tunability and reversibility of the FKBP-based PB transposon induction system. ( A ) FKBP-PBase-FKBP activity is tunable. Cells were transfected with donor and helper plasmids (FKBP-PBase-FKBP) and subjected to various concentrations of Shld1: 0 nM, 8 nM, 40 nM, 200 nM and 1 μM. The normalized number of puromycin-resistant colonies observed is plotted versus the Shld1 concentration for four cell lines. ( B ) Shld-1 induction is reversible. Fluorescent images are taken at various timepoints after induction with 1uM Shld1 or removal of Shld1 from transfected HEK293T cells. The white scale bar equals 50 μm. The red arrows indicate the time points of measurement. Bright field and fluorescent images were shown at top and bottom panel respectively. ( C ) Quantification of the reversibility of Shld-1 induction. The cells imaged in panel B were quantified by FACS. The normalized percentage of YFP positive cells is plotted at each time point for four cell lines.

Article Snippet: To select cells in which the PB transposon transposed, cells were trypsinized 2 days after transfection and cultured on 10 cm dish (Corning) with 10 ml fresh media containing puromycin (1 μg/ml).

Techniques: Activity Assay, Transfection, Concentration Assay, FACS