User:Cindy54683/DC Research

This page will be used for dendritic cell research!

What to use for DCs (treated with different chemokines, LPS, poly(I:C), etc with internalization mechanisms including phagocytosis, receptor-mediated endocytosis, and pinocytosis

Clignan 2006

Reverse Transcriptase - PCR (RNA extracted with isolation reagent, TRI REAGENT)

reverse transcribed with cDNA-->gel-->stained--> look @ band intensities

cells preincubated 10 min, FITC-DEX + chemokine cocurrently 2 min --> ICE (ENDOCYTOSIS)

FITC-DEX uptake BC1 cells treated w/ or w/o IL-4 or IFN-γ 48 hours

FITC-DEX added cocurrently with no chemokine or CCL3

Raiber 2010(Inhibitors to APC via mannose receptors)

Receptor Mediated endocytosis by mannose receptors (neomannosylated BSA)

Used TMR (fluoresence)

pepstatin=inhibitor of aspartic proteinases

Platt 2010

Receptor mediated endocytosis

1uM fluoresebrite yellow green carboxylated latex microspheres coated overnight with mouse igG or BSA before incubation

West 2004

GFP-actin amplified by PCR from PEGFP-actin vector -EcoRI site at both 5' and 3' ends using oligonucleotides

FITC-DEX 10 min 37C (MACROPINOCYTOSIS)

Yanagawa 2003

FITC-DEX or FITC-albumin???

Kikuchi 2005

FITC-DEX, FITC-BSA, Mann-FITC-BSA, S. aureus, FITC-E. coli

FITC-Dex uptake by BC1 cells treated with or without IL-4 or IFN-γ for 48 hours.

FITC-Dex was added cocurrently with no chemokine or CCL3

Segura 2009

Different cross-presentation pathways in steady-state and inflammatory dendritic cells

OVA conjugated to ALEXA488 30 min

What we currently have

Receptor-mediated endocytosis : FITC-Dextran

Pinocytosis: FITC-BSA

Phagocytosis: Polystyrene-coated beads

What is our concern?

BSA may be internalized by receptors???

OVA is internalized by mannose receptors on DC (Raiber 2010)) Measuring transfection efficiency for gene delivery

GFP Transfection efficiency (this is what Connie is using???...similar??)

ratio of cells expressing GFP to the total cell number.

Because the mammalian cell membrane is impenetrable to large molecules (e.g. DNA) that carry an electrical charge... researchers have...

developed carrier molecules

viral vectors

poking holes in membrance

carrier molecules

4 decades ago coating DNA with DEAE-dextran was found to allow this to get past the cell membrane

1970s: transfect DNA using calcium phosphate less toxic than DEAE-dextran

we are now looking at lipid-based reagents to offer greater ease of use and efficiency with lower toxicity

Vocab

mDC have a high density of CCR7 and this helps facilitate migration to regional lymph nodes

upon maturation DC downregulates CCR1 and CCR5 which are receptors for CCL3 CCL 19 and CCL21 are ligands for CCR 7...

mDC use CCR7 to migrate and stimulate naive T-cells, go to T-cell zones and lymph nodes

iDC have a high density of CCR1 and CCR5 which are receptors for CCL3 CCL3 is secreted by endothelial cells, epithelial cells, and leukocytes...

This recruits iDC and other immune cells to the site of inflammation

research cell transfection efficiency stuff... Localised transfection - we have developed a way of targeting DNA, containing gene network constructs, to locations within a cell population. Cells can thus be transfected to micrometer resolution using magnetic beads (Isalan

GFP... flow cytometry

Beta-gal (Lac-Z)- promoter linked to lacZ gene --> beta galactosidase --> blue color

luciferase assay

EGFP-luciverase fusion vector (clontech,BD biosci) measure % + for GFP absolute number light units/well

References

Gothelf 2010 Duration and level of transgene expression after gene electrotransfer to skin in mice. Luciferase plasmid was injected into mice, area electroporated, at different time points animals were anesthetized hair removed and injected with luciferin and relative luminescence was measured by bioimaging by Optix MX-2 Time Domain Optical Imaging.

Raiber 2010 Raiber et. al synthesied an inhibitor/carrier conjugate MPC6 which targets aspartic proteinase inhibitor pepstatin. They chose to use tetramethyl rhodamine (TMR) to allow efficient tracking of the conjugate. TMR allowed for multiparameter analysis in combination

Quantitative Proteomics Reveals Subset- Viral Recognition in Dendritic Cells Dendritic Cell Subsets Digested: RNA Sensing Makes the Difference!

Improving vaccines by targeting antigens to dendritic cells Cross-priming in health and disease