Arichards:Notebook/MeetingWithKota160317

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Meeting with Kota Kaneko 3/17/2016

Kota performed a FACS experiment on Wednesday 3/16/2016 on a single mouse.

<![if !supportLists]>·         <![endif]>Perfused liver with PBS to flush RBCs and platelets via portal vein

<![if !supportLists]>·         <![endif]>Perfused with collagenase I and IV and dissociated with razor

<![if !supportLists]>·         <![endif]>Spun down hepatocytes at 50 g for 5 min

<![if !supportLists]>·         <![endif]>Stained with Hoerst and PI with Verapamil (to block calcium channel, prevent pumping out of Hoerst)

<![if !supportLists]>o   <![endif]>Live cells stain with Hoerst but not PI

<![if !supportLists]>o   <![endif]>Dead cells stain for both

<![if !supportLists]>·         <![endif]>Sorted at Sanford HESSCF on FACScanto:

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The Feng group has a variety of livor tumor models:

<![if !supportLists]>·         <![endif]>Albumin-Cre mice (B6.Cg-Tg(Alb-cre)21Mgn/J) are crossed with a variety of floxed strains

<![if !supportLists]>o   <![endif]>Crossed with flox-stop GFP reporter to validate specificity of knockout

<![if !supportLists]>o   <![endif]>Saw GFP in hepatocytes only

<![if !supportLists]>·         <![endif]>Floxed PTEN and floxed Ptpn11 (SHP2), ~2 kb deletion of the promoter

<![if !supportLists]>o   <![endif]>Mouse Pten is on <a href="http://www.ensembl.org/Mus_musculus/Location/View?db=core;g=ENSMUSG00000013663;r=19:32757497-32826160">Chromosome 19: 32,757,497-32,826,160</a> forward strand.

<![if !supportLists]>o   <![endif]>Mouse Ptpn11 is on <a href="http://www.ensembl.org/Mus_musculus/Location/View?db=core;g=ENSMUSG00000043733;r=5:121130533-121191397">Chromosome 5: 121,130,533-121,191,397</a> reverse strand

<![if !supportLists]>·         <![endif]>DEN (Diethylnitrosamine) is also used to generate liver tumors

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