Journal: PLoS ONE

Article Title: Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation – An Appraisal

doi: 10.1371/journal.pone.0097443

Figure Lengend Snippet: Shown is a representative example of the procedure using the well-transfectable MM cell line JJN-3. This standard column purification has now been performed hundreds of times in our laboratory and is also easily applicable for MM cell lines INA-6, KMS-11, L-363, MM.1S and U-266 . a) Cell culture one day after electroporation with expression plasmids for enhanced green fluorescent protein (EGFP) and CD4Δ, showing about a quarter of cells strongly positive for EGFP. b)-e) Enrichment of strongly transfected cells by selection for CD4 surface expression (CD4 MicroBead column selection). b) Column runthrough of cell culture shown in a). Of note is the similar look with a), but with depletion of the strongest transfected cells in b). c) Column eluate of the cell culture shown in a). Untransfected cells (EGFP- and CD4Δ-negative) have effectively been removed, but the column procedure tends to retain significant amounts of dead cells (EGFP-negative, PI-positive). d) Floating fraction of the column eluate as shown in c) after “density gradient” (more properly: density step) treatment using OptiPrep, consisting mostly of viable and strongly transfected cells. e) Pelleted fraction of the column eluate as shown in c) after “density gradient” treatment using OptiPrep, consisting mostly of debris. f) Removal of debris by OptiPrep treatment from the cell culture as shown in a) without prior column separation, leaving two main fractions which are either EGFP-negative, or distinctly EGFP-positive. See Methods section for further details.

Article Snippet: For simple removal of debris and dead cells the cell cultures at either day 1 or day 2 post-electroporation were pelleted and resuspended in a mixture of OptiPrep (a 60% solution of iodixanol; Progen Biotechnik, Heidelberg, Germany) with full medium (2.5 ml medium + 0.75 ml OptiPrep).

Techniques: Purification, Cell Culture, Electroporation, Expressing, Transfection, Selection

Journal: PLoS ONE

Article Title: Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation – An Appraisal

doi: 10.1371/journal.pone.0097443

Figure Lengend Snippet: INA-6-EGFP cells were electroporated with a solution containing a stealth siRNA targeting EGFP as well as an expression plasmid for CD4Δ. One day post-electroporation one half of the cell culture was purified according to the column procedure (red curves, also see Fig. 1b)–e)), whereas the other half only underwent debris removal with OptiPrep (blue curves, also see Fig. 1f)). Purified cells were further cultured and FACS-analysed for EGFP expression at the times indicated. Only the live cell fraction (as demarcated in the forward/sideward scatter) was analysed and plotted against similarly treated INA-6-EGFP cells (green curves) transfected with a non-EGFP targeting siRNA. Knockdown efficiency was essentially identical in strength and over time between both purification approaches. One representative experiment from a total of three is shown.

Article Snippet: For simple removal of debris and dead cells the cell cultures at either day 1 or day 2 post-electroporation were pelleted and resuspended in a mixture of OptiPrep (a 60% solution of iodixanol; Progen Biotechnik, Heidelberg, Germany) with full medium (2.5 ml medium + 0.75 ml OptiPrep).

Techniques: Expressing, Plasmid Preparation, Electroporation, Cell Culture, Purification, Transfection, Knockdown

Journal: PLoS ONE

Article Title: Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation – An Appraisal

doi: 10.1371/journal.pone.0097443

Figure Lengend Snippet: Transfection of AMO-1 cells across a range of voltages using an expression vector for EGFP (pEGFP-N3) and a stealth siRNA against ERK2 (stERK2) in the electroporation mixture. Top panel: increases of the fractions of EGFP-expressing as well as of dead cells with higher voltages (top row). Cells taken in culture after OptiPrep-mediated debris removal reflect only the increase in transfection efficiency for the EGFP expression plasmid (bottom row). Middle panel: purified AMO-1 cells (those shown in the upper panel, bottom row) after culture for another 4 days. Top row: EGFP expression. Bottom row: annexin V-PromoFluor 647/PI staining. Even for the highest voltage used (320 V) the purified live cell fraction did not fare worse in subsequent culture than cells electroporated under milder conditions. Bottom panel: Western analysis of ERK2 knockdown at days 3 and 5 post-electroporation from the same cultures from which the FACS panels were derived. Efficient siRNA-mediated ERK2 knockdown was achieved at voltages significantly lower than required for the best levels of plasmid electroporation. However, a lower limit for successful knockdown was reached between the settings for 160 and 200 V. Shown is a representative experiment of two complete sets (Western blotting included). Anti-ERK1/2 antibody: CST.

Article Snippet: For simple removal of debris and dead cells the cell cultures at either day 1 or day 2 post-electroporation were pelleted and resuspended in a mixture of OptiPrep (a 60% solution of iodixanol; Progen Biotechnik, Heidelberg, Germany) with full medium (2.5 ml medium + 0.75 ml OptiPrep).

Techniques: Transfection, Expressing, Plasmid Preparation, Electroporation, Purification, Staining, Western Blot, Knockdown, Derivative Assay

Journal: PLoS ONE

Article Title: Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation – An Appraisal

doi: 10.1371/journal.pone.0097443

Figure Lengend Snippet: Left-hand panel: MM cell lines were electroporated with an expression vector for EGFP (pEGFP-N3; 10 µg/ml) and stealth siRNAs against either ERK2 (stERK2; 3 µM) or against no specific target (control; 3 µM). The FACS-measurements represent the cell cultures at day 1 post-electroporation after debris removal with OptiPrep. Right-hand panel: Knockdown of ERK2 and intrinsic levels of phospho-ERK2 (cells from the cultures represented on the left were harvested at day 3 post-electroporation for Western blotting). Good knockdown of ERK2 and lowered levels of phospho-ERK2 were found for all four MM cell lines tested. Shown is a representative experiment of two complete sets (Western blotting included). Anti-ERK1/2 antibody: Santa Cruz Biotechnology.

Article Snippet: For simple removal of debris and dead cells the cell cultures at either day 1 or day 2 post-electroporation were pelleted and resuspended in a mixture of OptiPrep (a 60% solution of iodixanol; Progen Biotechnik, Heidelberg, Germany) with full medium (2.5 ml medium + 0.75 ml OptiPrep).

Techniques: Expressing, Plasmid Preparation, Control, Electroporation, Knockdown, Western Blot

Journal: PLoS Biology

Article Title: Secretion and Signaling Activities of Lipoprotein-Associated Hedgehog and Non-Sterol-Modified Hedgehog in Flies and Mammals

doi: 10.1371/journal.pbio.1001505

Figure Lengend Snippet: (A) Density of human Shh secreted by HeLa cells in the absence or presence of fetal bovine serum (FBS), analyzed by Optiprep density gradient centrifugation, and Western blotting (WB). HeLa cells transfected with Shh were grown in serum-free medium or in the presence of 10% FBS, and equal volumes of supernatants analyzed. Colors indicate fractions corresponding to bovine Very Low-, Low-, and High-Density Lipoproteins (VLDL, LDL, and HDL) . (B) Density of non-lipid-modified Shh-N C24S , analyzed by Optiprep density gradient centrifugation and WB. Supernatants were derived from HeLa cells transfected with Shh-N C24S and grown in the presence of FBS. (C) Shh levels in cell lysates and supernatants derived from HeLa cells transfected with Shh, grown in serum-free medium supplemented with individual human lipoprotein classes. Equal protein amounts (cell lysates) or volumes (supernatants) were analyzed. (D) Density of Shh in HeLa cell supernatants shown in (C), analyzed by Optiprep density gradient centrifugation and WB. Colors indicate fractions corresponding to human VLDL, LDL, and HDL . (E) Co-Immunoprecipitation (Co-IP) of secreted Shh with different lipoprotein classes, analyzed by WB. Supernatants were derived from HeLa cells transfected with Shh or Shh-N C24S , grown in serum-free medium supplemented with individual human lipoproteins classes. (F) Shh levels in supernatants derived from MIA PaCa-2 cells grown in serum-free medium supplemented with individual human lipoprotein classes. Equal volumes were used for WB. (G) Density of Shh in MIA PaCa-2 cell supernatants shown in (F), analyzed by Optiprep density gradient centrifugation and WB. (H) Density of Shh in supernatants from Shh-expressing HeLa cells grown in serum-free medium supplemented with hemolymph from Drosophila larvae, analyzed by Optiprep density gradient centrifugation and WB. Purple indicates fractions corresponding to Drosophila Lpp.

Article Snippet: Isopycnic density centrifugation in Optiprep gradients (Fresenius Kabi) and KBr gradients was performed as described , .

Techniques: Gradient Centrifugation, Western Blot, Transfection, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing

Journal: PLoS Biology

Article Title: Secretion and Signaling Activities of Lipoprotein-Associated Hedgehog and Non-Sterol-Modified Hedgehog in Flies and Mammals

doi: 10.1371/journal.pbio.1001505

Figure Lengend Snippet: (A) Hh levels in hemolymph and whole extracts of wild-type and hh TS larvae at restrictive temperature, analyzed by WB. Hemolymph loading control is a secreted GFP expressed from the tubulin promoter. (B) Hemolymph Hh levels in wild-type and disp mutant larvae, analyzed by WB. Loading control is Cv-d. (C) Density of Hh in hemolymph of wild-type and Lpp RNAi larvae, analyzed by Optiprep density gradient centrifugation and WB. Equal amounts of hemolymph (normalized by protein) were analyzed. (D) Hemolymph Hh levels in larvae overexpressing Hh in imaginal discs with en105 -GAL4, analyzed by WB. Loading control is Cv-d. (E) Density of hemolymph Hh in larvae overexpressing Hh in imaginal discs, analyzed by Optiprep density gradient centrifugation and WB. (F) Density of Hh lipid modification mutants (Hh C85S , Hh-N, Hh-N C85S ), secreted into the hemolymph from the fat body (FB) with lpp -GAL4, analyzed by Optiprep density gradient centrifugation and WB.

Article Snippet: Isopycnic density centrifugation in Optiprep gradients (Fresenius Kabi) and KBr gradients was performed as described , .

Techniques: Mutagenesis, Gradient Centrifugation, Modification

Journal: PLoS Biology

Article Title: Secretion and Signaling Activities of Lipoprotein-Associated Hedgehog and Non-Sterol-Modified Hedgehog in Flies and Mammals

doi: 10.1371/journal.pbio.1001505

Figure Lengend Snippet: (A) Size of lipoprotein-associated and lipoprotein-free Shh secreted from HeLa cells grown in the presence or absence of FBS. Supernatants were fractionated by gel filtration chromatography on a Superdex 200 column, and fractions analyzed by WB. Molecular weight standards are indicated. Note that large mammalian lipoproteins run in the void volume of these columns. (B) Hemolymph Hh levels in Drosophila larvae ectopically expressing Hh in the fat body, with or without concomitant Lpp RNAi, analyzed by WB. (C) Density of hemolymph Hh produced in the fat body of control or Lpp RNAi larvae, analyzed by Optiprep density gradient centrifugation and WB. (D) Size of Lpp-associated and Lpp-free hemolymph Hh ectopically produced in the fat body of control or Lpp RNAi larvae, analyzed by gel filtration chromatography as described in (A). (E) Cholesterol modification status of low- and high-density forms of Drosophila Hh. Supernatant from Hh-producing S2 cells grown in the presence of FBS and 3 H-cholesterol was fractionated by KBr density gradient centrifugation, and subsequently analyzed by both WB and in-gel fluorography. (F) Hydrophobicity of low- and high-density forms of Hh. Supernatants from Hh-producing S2 cells grown in the presence of serum were fractionated by KBr density gradient centrifugation; fractions 1 and 8 were analyzed by Triton X-114 phase separation and WB. (G–I) Hydrophobicity of Shh and Hh produced in the presence or absence of lipoproteins, assessed by Triton X-114 phase separation and WB. (G) Lysates and media from HeLa cells expressing Shh and grown in the presence or absence of FBS. (H) Hemolymph from control and Lpp RNAi larvae. (I) Hemolymph from control and Lpp RNAi larvae ectopically producing Hh in the fat body. Note that aqueous phase Hh/Shh has a lower electrophoretic mobility than detergent phase Hh/Shh.

Article Snippet: Isopycnic density centrifugation in Optiprep gradients (Fresenius Kabi) and KBr gradients was performed as described , .

Techniques: Filtration, Chromatography, Molecular Weight, Expressing, Produced, Gradient Centrifugation, Modification