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protocol_69.ann
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T1 Action 27 30 Add
E1 Action:T1 Acts-on:T3 Site:T5
T2 Amount 39 42 2ml
T3 Reagent 31 38 Trypsin
T4 Action 54 59 shake
E2 Action:T4 Acts-on:T5
T5 Location 60 65 plate
R1 Measure Arg1:T3 Arg2:T2
T6 Action 67 70 Add
E3 Action:T6 Acts-on:T8
T7 Amount 71 74 8ml
T8 Reagent 75 96 ES medium without LIF
R2 Measure Arg1:T8 Arg2:T7
T9 Reagent 111 118 trypsin
T10 Action 100 110 inactivate
E4 Action:T10 Acts-on:T9
T11 Action 120 130 Centrifuge
E5 Action:T11
T12 Speed 134 140 500rpm
T13 Time 145 150 10min
R3 Setting Arg1:E5 Arg2:T12
R4 Setting Arg1:E5 Arg2:T13
T14 Action 152 155 Add
E6 Action:T14 Acts-on:T16 Site:T18
T15 Amount 156 159 8ml
T16 Reagent 163 172 EB medium
T17 Size 181 185 10cm
T18 Location 186 196 petri-dish
R5 Measure Arg1:T18 Arg2:T17
R6 Measure Arg1:T16 Arg2:T15
T19 Action 224 233 resuspend
E7 Action:T19 Acts-on:T20 Using:T22 Count:T25 Using2:T24 Using3:T138
T20 Reagent 234 240 pellet
T21 Amount 244 247 5ml
T22 Reagent 248 257 EB medium
T23 Amount 261 264 5ml
T24 Device 265 272 pipette
T25 Numerical 290 297 5 times
R7 Measure Arg1:T22 Arg2:T21
T26 Action 299 303 Take
E8 Action:T26 Acts-on:T28
T27 Amount 304 309 100ul
T28 Reagent 310 325 cell suspension
T29 Action 330 336 dilute
E9 Action:T29 Acts-on:T28
T30 Amount 342 345 1ml
R9 Measure Arg1:T28 Arg2:T27
R10 Setting Arg1:E9 Arg2:T30
T31 Action 354 358 Take
E10 Action:T31 Count:T32 Acts-on:T33
T32 Numerical 359 377 appropriate number
T33 Reagent 381 386 cells
T34 Action 391 397 dilute
E11 Action:T34 Acts-on:T33
T35 Concentration 403 418 1×10^6 cells/ml
R11 Setting Arg1:E11 Arg2:T35
T36 Action 427 437 re-suspend
E12 Action:T36 Using:T38 Count:T39 Using2:T140
T37 Size 441 444 5ml
T38 Device 445 452 pipette
T39 Numerical 470 477 4 times
T40 Action 479 483 Seed
E13 Action:T40 Acts-on:T42 Site:T43
T41 Amount 484 487 2ml
T42 Reagent 491 496 cells
T43 Location 497 501 dish
T44 Action 503 507 Save
E14 Action:T44 Acts-on:T46 Using:T47
T45 Amount 508 514 2×10^6
T46 Reagent 515 520 cells
T47 Method 525 539 RNA extraction
T48 Action 541 549 Transfer
E15 Action:T48 Acts-on:T49 Site:T51
T49 Reagent 550 571 medium containing Ebs
T50 Size 575 579 50ml
T51 Location 580 585 tubes
T52 Action 597 600 add
E16 Action:T52 Acts-on:T54 Site:T55
T53 Amount 601 604 5ml
T54 Reagent 605 611 medium
T55 Location 615 621 dishes
T56 Action 638 649 put back in
E17 Action:T56 Acts-on:T55 Site:T57
T57 Device 650 659 incubator
T58 Action 661 665 Sink
E18 Action:T58 Acts-on:T59 Site:T60 Using:T61
T59 Reagent 666 671 cells
T60 Location 675 680 tubes
T61 Method 681 691 by gravity
T62 Time 696 700 5min
T63 Action 702 709 Discard
E19 Action:T63 Acts-on:T64
T64 Reagent 710 721 supernatant
T65 Action 723 726 Add
E20 Action:T65 Acts-on:T67 Site:T68
T66 Amount 727 730 5ml
T67 Reagent 731 737 medium
T68 Reagent 741 748 pellets
T69 Reagent 754 761 samples
T70 Action 750 753 Mix
E21 Action:T70 Acts-on:T69 Using:T71 Count:T72
T71 Method 762 774 by inverting
T72 Numerical 775 786 a few times
T73 Action 795 805 Distribute
E22 Action:T73 Acts-on:T74 Site:T75
T74 Reagent 806 813 samples
T75 Location 827 833 dishes
T76 Action 835 842 Prepare
E23 Action:T76 Acts-on:T77
T77 Reagent 843 872 gelatin coated chamber slides
T78 Action 883 886 Add
E24 Action:T78 Acts-on:T80 Site:T81
T79 Amount 887 892 1.5ml
T80 Reagent 896 902 medium
T81 Location 913 917 well
T82 Action 919 927 Transfer
E25 Action:T82 Acts-on:T85 Site:T87
T83 Amount 928 934 1 dish
T84 Modifier 938 942 day4
T85 Reagent 943 946 Ebs
T86 Size 952 956 15ml
T87 Location 957 961 tube
T88 Reagent 972 975 Ebs
T89 Location 998 1002 tube
T90 Method 1003 1013 by gravity
T91 Time 1018 1022 5min
T92 Action 976 980 sink
E26 Action:T92 Acts-on:T88 Using:T90 Site:T145
T93 Action 1024 1031 Discard
E27 Action:T93 Acts-on:T94
T94 Reagent 1032 1043 supernatant
T95 Action 1045 1048 Add
E28 Action:T95 Acts-on:T97 Site:T98
T96 Amount 1049 1053 10ml
T97 Reagent 1054 1063 EB medium
T98 Reagent 1069 1076 pellets
T99 Action 1078 1082 Seed
E29 Action:T99 Using:T101 Site:T102
T100 Amount 1083 1088 0.5ml
T101 Reagent 1092 1094 EB
T102 Location 1103 1107 well
T103 Action 1109 1115 Change
E30 Action:T103 Acts-on:T104
T104 Reagent 1116 1122 medium
T105 Action 1133 1137 suck
E31 Action:T105 Acts-on:T106
T106 Reagent 1138 1149 supernatant
T107 Action 1151 1158 replace
E32 Action:T107 Acts-on2:T106 Using:T108
T108 Reagent 1170 1179 EB medium
T109 Modifier 1164 1169 fresh
T110 Action 1181 1185 Take
E33 Action:T110 Count:T111 Acts-on:T112
T111 Numerical 1186 1187 2
T112 Location 1188 1193 wells
T113 Action 1234 1238 Suck
E34 Action:T113 Acts-on:T114
T114 Reagent 1243 1249 medium
T115 Action 1261 1264 Fix
E35 Action:T115 Using:T117
T116 Concentration 1270 1272 4%
T117 Reagent 1273 1276 PFA
T118 Time 1281 1286 30min
T119 Temperature 1288 1290 RT
T121 Action 1303 1307 Wash
E36 Action:T121 Using:T123
T122 Amount 1316 1319 2ml
T123 Reagent 1323 1326 PBS
T124 Action 1328 1332 Wash
E37 Action:T124 Using:T126
T125 Amount 1341 1344 2ml
T126 Reagent 1348 1351 PBS
T127 Action 1353 1357 Wash
E38 Action:T127 Using:T129
T128 Amount 1366 1369 2ml
T129 Reagent 1373 1376 PBS
T130 Action 1378 1383 Store
E39 Action:T130 Acts-on:T131 Site:T132
T131 Reagent 1384 1391 samples
T132 Reagent 1395 1398 PBS
T133 Temperature 1402 1405 4°C
R12 Measure Arg1:T38 Arg2:T37
R13 Measure Arg1:T42 Arg2:T41
R14 Measure Arg1:T46 Arg2:T45
R15 Measure Arg1:T51 Arg2:T50
R16 Measure Arg1:T54 Arg2:T53
R17 Setting Arg1:E18 Arg2:T62
R18 Measure Arg1:T67 Arg2:T66
R19 Measure Arg1:T80 Arg2:T79
R20 Mod-Link Arg1:T85 Arg2:T84
R21 Measure Arg1:T85 Arg2:T83
R22 Measure Arg1:T87 Arg2:T86
R23 Setting Arg1:E26 Arg2:T91
R24 Measure Arg1:T97 Arg2:T96
R25 Measure Arg1:T101 Arg2:T100
R26 Mod-Link Arg1:T108 Arg2:T109
R27 Measure Arg1:T117 Arg2:T116
R28 Setting Arg1:E35 Arg2:T118
R29 Setting Arg1:E35 Arg2:T119
R30 Measure Arg1:T123 Arg2:T122
R31 Measure Arg1:T126 Arg2:T125
R32 Measure Arg1:T129 Arg2:T128
R33 Setting Arg1:E39 Arg2:T133
T134 Modifier 47 53 gently
R34 Mod-Link Arg1:E2 Arg2:T134
T135 Modifier 176 180 each
R35 Mod-Link Arg1:T18 Arg2:T135
T136 Method 203 215 centrifuging
T137 Modifier 217 223 Gently
R36 Mod-Link Arg1:E7 Arg2:T137
T138 Method 274 285 up and down
R8 Setting Arg1:T24 Arg2:T23
T139 Modifier 420 426 Gently
R37 Mod-Link Arg1:E12 Arg2:T139
T140 Method 454 465 up and down
T141 Modifier 587 596 Carefully
R38 Mod-Link Arg1:E16 Arg2:T141
T142 Modifier 622 633 immediately
R39 Mod-Link Arg1:E16 Arg2:T142
T143 Modifier 817 826 different
R40 Mod-Link Arg1:T75 Arg2:T143
T144 Modifier 908 912 each
R41 Mod-Link Arg1:T81 Arg2:T144
T145 Location 988 994 bottom
R42 Meronym Arg1:T145 Arg2:T89
T146 Modifier 1098 1102 each
R43 Mod-Link Arg1:T102 Arg2:T146
T147 Modifier 1250 1259 carefully
R44 Mod-Link Arg1:E34 Arg2:T147