ࡱ> 9;8 bjbj̚̚ 0&,E,=0000000:,d  0ER 0 d0dD0E  n:   Split ES cells 0.1% gelatin 0.125% trypsin ES cell media Standard cell media Coat dish in gelatin, let sit at RT for ~2 minutes, then aspirate solution Wash ES cells with PBS Add 0.125% trypsin let stand at RT for 3 minutes (4ml for 10cm plate, 0.5ml for 6-well) Add 3.5/8 ml of standard media to quench trypsin Pipette up and down 10x tighly against bottom of plate to break clumps and bring to single cell suspensionimportant to avoid differentiation and death, especially if growing off of MEFs Remove enough cells to split 1:6. Target cell # is 1 million/25 cm^2. If needed scale this number higher or lower to get ~90% confluency 2 days later. Discard remainder. Spin down cells at 1000rpm for 5 min R/S cells in ES media plus LIF. 2ml for 6-well, 10ml for 10cm. For happiest cells, change media every day and split when cells reach ~90% confluence. Differentiation into Embryoid Bodies: From ESC plate make suspension of single cells, preplate 35 minutes to get rid of MEFs, and dilute to 1 million cells per 10 mil, in 10% Fetal calf serum, glutamine, Pen/Strep, NAP, NEAA, DMEM. Plate cells onto a non-TC treated petri dish. Can also coat here with 0.5-1% agarose in DMEM to prevent EB attachment. If not coating, blow attached colonies off bottom of plate each day. At day 4 split 1 to 4. Note: XCI can be Counting ES cells with a hemacytometer 1) put 10ul of cell suspension at bottom of 1.5ml eppendorf 2) remove from hood and add 10ul of trypan blue stain 3) add 20ul cell suspension to clean hemacytometer under cover slip 4) count 3-4 fields, take average 5) cells per ml is (count) * (2 * 10^4) ES Media DMEM high glucose plus sodium pyruvate 0.1mM non-essential AA (100x stock from Gibco) 0.1mM B-Mercaptoethanol (4ul of reagent from sigma or 1000x dilution of our stock) P/S antibiotics L-Glu (100x from Gibco) 500U/ml LIF if growing on MEFs, otherwise 1000U/ml LIF (Gibco/Chemicon) 15% Defined FBS, ES qualified, from Gibco/Hyclone (SH30070) Standard Media DMEM high glucose plus sodium pyruvate 0.1mM B-Mercaptoethanol (4ul of reagent from sigma or 1000x dilution of our stock) P/S antibiotics L-Glu (100x from Gibco) 10% FBS (non-qualified) ES cell trypsin 0.125% trypsin (diluted from 0.25% trypsin with PBS) 0.1% gelatin 0.5g of gelatin in 500ml of PBS, autoclave Prepping ES cell DNA -Put 1ml of trypsinized cell suspension into eppendorf, spin out media, add 500ul of ES lysis buffer supplemented 1:100 with proteinase K (in fridge) 100 mM Tris-HCl, pH 8.1, 5mM EDTA, pH 8.0, 200mM NaCl, 0.2% SDS. 50mls: 1M Tris 8.1 5 ml 500mM EDTA 0.5 ml 5M NaCl 2 ml 10% SDS 1 ml H20 41.5 ml -Put lysis mixture at 56 C in dry heat overnight -Next day add 1ml of ice cold ethanol -Rotate in cold room for 15 minutes -Spin in centrifuge at top speed for 5 minutes -Aspirate off Ethanol/lysis buffer mixture -Add 500 ul of Buffer TE to DNA pellet and centrifuge to loosen pellet -Put resuspended pellet at 56 C dry heat overnight to let DNA dissolve Sex determination of ES cells from Current Protocols in Mol. Bio., Chapter 23.4  HYPERLINK "http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/article/mb2304/current/html#mb2304-prot-0002" http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/article/mb2304/current/html#mb2304-prot-0002 Primers (Kunieda et al., 1992): SRY2: TCTTAAACTCTGAAGAAGAGAC SRY4: GTCTTGCCTGTATGTGATGG NDS3: GAGTGCCTCATCTATACTTACAG NDS4: TCTAGTTCATTGTTGAGTTGC 35 cycles 30 sec 94 30 sec 50 30 sec 60 The SRY primers are derived from the sex-determining region of the Y chromosome. Male cells will show the 404-bp product; female cells will have no product. 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