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protocol_518.ann
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T3 Action 243 249 Pellet
E3 Action:T3 Acts-on:T1 Using:T2
T4 Action 324 330 remove
E4 Action:T4 Acts-on:T24
T5 Action 335 342 discard
E5 Action:T5 Acts-on:T24
T6 Action 360 363 Add
E6 Action:T6 Acts-on:T27
T7 Action 407 413 vortex
E7 Action:T7 Acts-on:T28
T8 Action 439 447 incubate
E8 Action:T8 Acts-on:T28
T9 Action 560 564;580 591 pull up and down
E9 Action:T9 Acts-on:T33 Count:T34 Using:T32
T10 Action 683 686 Add
E10 Action:T10 Acts-on:T37
T12 Action 836 846 Centrifuge
E12 Action:T12 Acts-on:T39
T13 Action 903 911 Transfer
E13 Action:T13 Acts-on:T42 Site:T43
T14 Action 943 953 Centrifuge
E14 Action:T14 Acts-on:T44
T15 Action 1029 1037 Transfer
E15 Action:T15 Acts-on:T47 Site:T48
T16 Action 1088 1091 Add
E16 Action:T16 Acts-on:T51 Site:T52
T17 Action 1138 1148 centrifuge
E17 Action:T17
T18 Action 1184 1191 Discard
E18 Action:T18 Acts-on:T55
T19 Action 1209 1216 Suspend
E19 Action:T19 Acts-on:T56 Using:T58 Creates:T59
T20 Action 1296 1300 keep
E20 Action:T20 Acts-on:T59
T21 Action 1375 1381 stored
E21 Action:T21
T1 Reagent 254 269 harvested cells
T2 Method 273 287 centrifugation
T22 Speed 291 299 ~800 x g
T23 Time 304 312 1 minute
R1 Setting Arg1:E3 Arg2:T22
R2 Setting Arg1:E3 Arg2:T23
T24 Reagent 347 358 supernatant
T25 Amount 364 369 500µl
T26 Temperature 373 381 ice cold
T27 Reagent 382 398 SubCell Buffer-I
R3 Measure Arg1:T27 Arg2:T25
R4 Setting Arg1:E6 Arg2:T26
T28 Reagent 429 434 cells
T29 Temperature 451 454 ice
T30 Time 459 469 10 minutes
R5 Setting Arg1:E8 Arg2:T29
R6 Setting Arg1:E8 Arg2:T30
T31 Size 527 535 20 gauge
T32 Device 537 551 syringe needle
R7 Measure Arg1:T32 Arg2:T31
T33 Reagent 569 579 suspension
T34 Numerical 592 603 10-30 times
T35 Amount 687 692 250µl
T36 Concentration 693 695 3X
T37 Reagent 696 713 SubCell Buffer-II
R8 Measure Arg1:T37 Arg2:T36
R9 Measure Arg1:T37 Arg2:T35
T38 Action 756 759 mix
E2 Action:T38 Using:T11
T11 Method 763 772 inverting
T39 Location 851 855 tube
T40 Speed 859 865 700x g
T41 Time 870 880 10 minutes
R10 Setting Arg1:E12 Arg2:T40
R11 Setting Arg1:E12 Arg2:T41
T42 Reagent 916 927 supernatant
T43 Location 937 941 tube
T44 Reagent 954 965 supernatant
T45 Speed 969 978 12,000x g
T46 Time 983 993 15 minutes
R12 Setting Arg1:E14 Arg2:T45
R13 Setting Arg1:E14 Arg2:T46
T47 Reagent 1042 1053 supernatant
T48 Location 1082 1086 tube
T49 Amount 1092 1097 500µl
T50 Concentration 1098 1100 1X
T51 Reagent 1101 1118 SubCell Buffer-II
T52 Reagent 1126 1132 pellet
R14 Measure Arg1:T51 Arg2:T50
R15 Measure Arg1:T51 Arg2:T49
T53 Speed 1158 1168 12,000 x g
T54 Time 1173 1182 5 minutes
R16 Setting Arg1:E17 Arg2:T53
R17 Setting Arg1:E17 Arg2:T54
T55 Reagent 1196 1207 supernatant
T56 Reagent 1221 1241 mitochondrial pellet
T57 Amount 1245 1253 50-100µl
T58 Reagent 1256 1291 Working Mitochondria Storage Buffer
R18 Measure Arg1:T58 Arg2:T57
T59 Reagent 1305 1315 suspension
T60 Temperature 1319 1322 ice
R19 Setting Arg1:E20 Arg2:T60
T61 Temperature 1385 1388 ice
T62 Time 1399 1407 48 hours
R20 Setting Arg1:E21 Arg2:T61
R21 Setting Arg1:E21 Arg2:T62