The Immunology Edition

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troporation protocols at:

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A New Method to Generate Quadromas by Electrofu-sion and FACS Sorting Electro-FACS-fusion is a simple, rapid method for the generation of high-frequency hybrid-hybridomas (quadromas). This new technique uses fluorescence activated cell sorting (FACS) following electrofusion which eliminates the need for double resistant cell lines*. The two parent hybridomas are labeled with fluorescein isiothiocyanate (FITC) and tetramethylrho-damine isothiocyanate (TRITC) prior to the fusion. After the electrofusion, cells exhibit-ing dual fluorescence are selected by FACS. The fused cells can be directly plated in microplates for clonal growth.

Under optimum conditions, it was demon-strated that electrofusion of 2 x 105 cells resulted in 30 hybridomas in the mouse system and 10 hybridomas in the human system. These efficiencies were about 10 times higher than those by PEG fusion*. Electrofusion offers several distinct advan-tages over the use of PEG in the genera-tion of hybridomas. It is less labor intensive, eliminating the need for repeated washings, and may be more efficient in producing a large number of fused cells. In addition, fewer cells are required. It is very difficult to perform a successful PEG fusion with fewer than 106 cells, but electrofusion can theo-retically be performed with as few as two cells*. Cells undergoing electrofusion are subject to localized membrane breakdown; on the other hand, cell membranes under-going fusion with PEG are uniformly af-fected, which may cause a greater loss of cellular constituents*. Visualization of the fusion process makes the production of hybrids theoretically possible, even without the use of a selection system. Another potential application of this method is in the development of primary hybridomas. Mye-loma cells can be labeled with TRITC, and antigen-specific B-cells could be selected using the antigen labeled with FITC.

Kreutz, F. T. et al., A New Method to Generate Quadromas by Electrofusion and FACS Sorting, Volume 17, Number 3, 1998, Hybridoma

75%

80%

85%

90%

Viability Efficiency

BTXpress Buffer vs Invitrogen's Opti-MEM in

Human primary T-cell Transfection

BTXpress Buffer

Opti-MEM

BTXpress High Performance Electroporation Buffer Compared to OptiMem™ BTXpress buffer was compared to Invitrogen’s Opti-MEM® medium in the electroporation of primary human T cells. Cells were transfected with plasmid DNA and the viabilities and efficiencies were assessed. Cells transfected with BTXpress buffer yielded similar efficiencies as Invitrogen’s Opti-MEM medium, thus demonstrating that BTXpress high performance electroporation buffer is capable of achieving similar transfection efficien-cies when compared to higher priced competitor systems and electroporation reagents. Source: Yangbing Zhao, PhD. University of Pennsylvania. yangbing@exchange.upenn.edu

Electrofusion of a Weakly Immunogenic Neuroblastoma with Dendritic Cells Pro-duces a Tumor Vaccine

The absence of surface costimulatory molecules explains in part the lack of an effective anti-

tumor immune response in tumor-bearing animals, even though unique tumor antigens may

be presented by class I MHC. We determined that the immunogenicity of a murine neuroblas-

toma, Neuro-2a, which lacks surface costimulatory molecules, could be increased by electri-

cally induced fusion with dendritic cells. Electrofusion induced a higher level of cell fusion

than polyethylene glycol, and tumor/dendritic cell heterokaryons expressed high levels of

costimulatory molecules. While Neuro-2a was unable to induce the proliferation of syngeneic

or allogeneic T cells in vitro, fused cells were able to induce T cell responses both in vitro

and in vivo. When fused dendritic tumor cells were used as a cancer vaccine, immunized

mice were significantly protected from challenge with Neuro-2a. We propose

that electrofusion with patient-derived tumor and dendritic cells may provide a rapid means

to produce patient-specific tumor vaccines.

Note. Fusion of dendritic cells to tumor cells was determined by loading cells with either CMFDA or CMTMR, carrying out PEG mediated cell fusion or electrofusion (as described under Materials and Meth-

ods), and then analyzing the fused population of cells by flow cytometry. Cells that gave a positive fluores-

cence signal for both CMFDA and CMTMR were considered fused. This experiment is representative of three separate determinations which gave similar results. a Dye-loaded DC and tumor cells were mixed at

various ratios. The cells were then “fused” or not fused (see Materials and Methods for details) and ana-

lyzed by flow cytometry to determine the percentage of total cells fused. Percentage fusion was determined by subtracting the percentage of double-positive cells of the unfused cells (i.e., not exposed to PEG or elec-

tric current) from the double-positive cells that underwent the respective fusion procedure. b A total of 1x106

cells was used for PEG-mediated fusion experiments while a total of 5 x 105 cells was used for each elec-

trofusion. Rimas J. Orentas,1 Dennis Schauer, Qian Bin, and Bryon D. Johnson Department of Pediatrics, Section of Hematology–Oncology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226 Cellular Immunology 213, 4–13 (2001)

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1) http://www.umass.edu/vetimm/docs/Wagner_Hybridoma.pdf 2) Bartal, A. et al.,Methods of Hybridoma Formation,1987 3) Orentas, R. et al.,Electrofusion of a Weakly Immunogenic Neuroblastoma with Dendritic Cells Produces a Tumor Vaccine, Cellular Immunology 213, 4–13 (2001) 4) Karsten, U., Stolley, P., Walther, I., Papsdorf, G., Weber, S., Conrad, K., Pasternak, L., Kopp, J.,1988, Direct comparison of electric field-mediated and PEG-mediated cell fusion for the generation of antibody producing hybridomas. Hybridoma 7, 627-633 5) Radomska, H. et al., Mammalian Cell Fusion in an Electroporation Device, 1995, Journal of Immunological Methods 6) Kreutz, F. T. et al., A New Method to Generate Quadromas by Electrofusion and FACS Sorting, Volume 17, Number 3, 1998,

Hybridoma 7) Lentz, BR. Et al., Poly(ethylene glycol) (PEG)-mediated fusion between pure lipid bilayers: a mechanism in common with viral fusion and secretory vesicle release?, 1999, Molecular Membrane Biology 8) Lentz, Br et al., Polymer-induced membrane fusion: potential mechanism and relation to cell fusion events, 1994, Chem Phys Lipids

BTX Electrofusion is as easy as 1 - 2 - 3 !!

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WHERE YOU NEED

TO USE:

• In vivo elec-

trodes

• High throughput

transfections

• Transfection of a

difficult cell line

• Applications

support

• Access to a li-

brary of tested

protocols

BTX IS YOUR

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BTX/Harvard Apparatus has acquired the world-wide exclusive right to manufacture and sell, for research use, the full line of electroporation-based instruments acquired by Cellectis from CytoPulse in September of 2010

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1. An oscillating AC waveform pulse aligns cells to be fused

2. A DC waveform pulse is applied to fuse the cells 3. A final AC waveform pulse is briefly applied to hold

cells together during recovery post fusion

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Electro-Molecular Therapy using Adult Mesen-

chymal Stem Cells

Abstract— Clinically chemo-refractive types of cancers do not respond well to conventional therapies. To treat and enhance the efficacy of drug delivery for these cancers, we have devel-oped an in vitro model of a combination therapy using adult Mesenchymal stem cells. Adult Mesenchymal stem cells have been used for this study primarily because of their ability to home towards tumor cells, making the possibility to practice targeted tumor therapy more realistic. These cells, derived from Human adult bone marrow were subjected to high intensity, short duration (1200V/cm, 100µs), and low intensity, long duration (200V/cm, 40ms and 450V/cm, 25ms) pulses. The effect of these voltages on the viability and proliferation ability of these cells in the presence and absence of Bleomycin (FDA ap-proved chemodrug used for treating various cancers) indicate the possibility of transfer of this technique to clinical practice for effective electro-molecular targeted stem cell therapy. An analysis of the electrical energy applied vs. the viability illustrates a linear relationship. The dose curve exhibits a non-linear relationship. These results indicate that the high efficacy of MSC tar-geted combination therapy would provide efficient, economical, and en-hanced clinical benefit for many types of cancers which need alternate treat-ments.

Figure 3: Adult Mesenchymal Stem cells from a 42 year old male patient .

Figure 1: Cancer stem cells (CSCs) are cancer cells (found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. CSCs are therefore tumorigenic (tumor-forming), perhaps in contrast to other non-tumorigenic cancer cells

Electro-Molecular Therapy using Adult Mesen-chymal Stem Cells Kavitha Sankaranarayanan1, Raja Prabhu Ramachandran2,M Sriram Kumar2, V Madan Kumar2, S Vignesh2, V Malini2, J Chris Maria Renny1, Soma Guhathakurta1, K.M.Cherian1, Arutselvan Natarajan3, and Raji Sundararajan4

Proc. ESA Annual Meeting on Electrostatics 2010, Paper I3 1Frontier Lifeline, Chennai, India 2B.S. Abdur Rahman University, Chennai, India 3Stanford Medical School, CA, USA 4Purdue University, West Lafayette, IN, USA

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Enhancer 3000 includes Oscilloscope, Interface box, and Voltage probe. For detailed monitoring of elec-trical parameters, improve fusion and transfections

Microslides for fusion or electroporation applica-tions. Comes in 0.5 mm, 1.0 mm, 3.2 mm and 10 mm sizes

2-Needle Array electrode. Ideal for in vivo gene delivery in muscle or DNA vaccine applications

High Performance BTXpress Solution. To improve transfection viabil-ities and efficiencies for mammalian cell transfec-tions

Microject 1000 Max System for nuclear transfer, transgenics, IVT and cell injection

RNAi screening of the tyrosine kinome identifies

therapeutic targets in acute myeloid leukemia

Despite vast improvements in our understanding of cancer genetics, a large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of numerous types of cancer, but identification and validation of tyrosine kinase targets in cancer can be a time-consuming process. We report the establishment of an efficient, functional screening assay using RNAi technology to directly assess and compare the effect of individually targeting each member of the tyrosine kinase family. We demonstrate that siRNA screening can identify tyrosine kinase targets containing activating mutations in Janus kinase (JAK) 3 (A572V) in CMK cells and c-KIT (V560G) in HMC1.1 cells. In addition, this assay identifies targets that do not contain mutations, such as JAK1 and the focal adhesion kinases (FAK), that are crucial to the survival of the cancer cells. This technique, with additional develop-ment, might eventually offer the potential to match specific therapies with individual patients based on a functional assay.

Figure 2. Optimization of electroporation in CMK cells.

(A) CMK cells were incubated with a FITC-labeled siRNA molecule and left untreated or electroporated at 300 V, 100 µsec, 2 pulses. After 48 hours cells were

analyzed for FITC incorporation by flow cytometry. (B) CMK cells were treated as in panel A and stained with propidium idodide. Viability as measured by PI exclu-sion was determined by flow cytometry on a Guava Technologies flow cytometer. (C) CMK cells were incubated with 0, 500, or 1000 nM siRNA targeting GAPDH and electroporated as in panel A. After 48 hours cell lysates were subjected to immunoblot analysis for GAPDH and β-actin.

Jeffrey W. Tyner, Denise K. Walters, Stephanie G. Willis, Mary Luttropp, Jason Oost, Marc Loriaux, Heidi Erickson, Amie S. Corbin, Thomas O'Hare, Michael C. Heinrich, Michael W. Deininger and Brian J. Druker

Blood. 2008;111: 2238-2245

IgE-mediated mast cell degranulation and release

of vasoactive mediators induced by allergens elicits allergic responses. Although

G protein-coupled receptor (GPCR)-induced sig-

nals may amplify IgE-dependent degranulation, how GPCR signaling in mast cells

is regulated remains incompletely defined. We

investigated the role of regulator of G protein signaling (RGS) proteins in the

modulation of these pathways in human mast

cells. Several RGS proteins were expressed in mast cells including RGS13, which

we previously showed inhibited IgE-mediated

mast cell degranulation and anaphylaxis in mice. To characterize how RGS13 affects

GPCR-mediated functions of human mast cells,

we analyzed human mast cell lines (HMC-1 and LAD2) depleted of RGS13 by

specific small interfering RNA or short hairpin

RNA and HMC-1 cells overexpressing RGS13. Transient RGS13 knockdown in

LAD2 cells lead to increased degranulation to

sphingosine-1-phosphate but not to IgE-Ag or C3a. Relative to control cells, HMC-1

cells stably expressing RGS13-targeted short hairpin RNA had greater Ca2� mobilization in

response to several natural GPCR

ligands such as adenosine, C5a, sphingosine-1-phosphate, and CXCL12 than wild-type cells. Akt

phosphorylation, chemotaxis, and

cytokine (IL-8) secretion induced by CXCL12 were also greater in short hairpin RGS13-HMC-1

cells compared with control.

RGS13 overexpression inhibited CXCL12-evoked Ca2� mobilization, Akt phosphorylation and

chemotaxis. These results suggest

that RGS13 restricts certain GPCR-mediated biological responses of human mast cells. The

Journal of Immunology, 2008, 181:

7882–7890.

Figure 7 Overexpression and localization of RGS13 in HMC-1 cells. A, Two individual transfectants expressing HA-RGS13 (OE1 and OE2) were ana-lyzed by immunoblotting with anti-RGS13 Ab and compared with cells transfected with empty vector (vec). Ectopically expressed RGS13 could be differ-entiated from endogenous protein by its different migration due to the fusion of 3 HA tags to RGS13. B, Localization of HA-RGS13 by immunocytochem-istry. Vector- or HA-RGS13-expressing cells were stained with anti-HA (B) or anti-RGS13 Ab (C) fol-lowed by microscopy (confocal images in C). In C, RGS13 staining is green; nuclei are stained �in blue with 4 -6-diamidino-2-phenylindole. Geetanjali Bansal, Jeffrey A. DiVietro, Hye Sun Kuehn, Sudhir Rao, Karl H. Nocka, Alasdair M. Gilfillan and Kirk M. Druey

RGS13 Controls G Protein-

Coupled Receptor-Evoked Re-

sponses of Human Mast Cells

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