protocol_236.ann

T1	Action 60 64	Grow
E1	Action:T1 Creates:T2 Site:T4
T2	Reagent 65 70	cells
T3	Size 74 79	18 mm
T4	Location 80 99	round #1 coverglass
T5	Size 105 112	12-well
T6	Reagent 113 131	cell culture plate
R1	Measure Arg1:T6 Arg2:T5	
R2	Measure Arg1:T4 Arg2:T3	
R3	Meronym Arg1:T4 Arg2:T6	
T7	Action 133 141	Aspirate
E2	Action:T7 Acts-on:T8
T8	Reagent 142 155	growth medium
T9	Action 161 165	wash
E3	Action:T9 Using:T12 Acts-on:T8
T10	Amount 171 175	1 mL
T11	Concentration 179 181	1X
T12	Reagent 182 185	PBS
R4	Measure Arg1:T12 Arg2:T11	
R5	Measure Arg1:T12 Arg2:T10	
T13	Action 190 193	fix
E4	Action:T13 Acts-on:T15 Using:E6
T14	Action 198 210	permeabilize
E5	Action:T14 Acts-on:T15 Using:E6
T15	Reagent 211 216	cells
T16	Action 218 221	add
E6	Action:T16 Acts-on:T18
T17	Amount 222 226	1 mL
T18	Reagent 230 280	methanol-acetic acid (MeOH-AcOH) fixation solution
R6	Measure Arg1:T18 Arg2:T17	
T19	Action 282 290	Incubate
E7	Action:T19 
T20	Temperature 294 310	room temperature
T21	Time 315 325	10 minutes
R7	Setting Arg1:E7 Arg2:T20	
R8	Setting Arg1:E7 Arg2:T21	
T22	Reagent 327 332	Cells
T23	Action 340 346	stored
E8	Action:T23 Acts-on:T22 Site:T25
T24	Temperature 350 361	+2 to +8 °C
T25	Reagent 365 374	MeOH-AcOH
T26	Time 381 389	48 hours
R9	Setting Arg1:E8 Arg2:T24	
R10	Setting Arg1:E8 Arg2:T26	
T27	Action 530 534	warm
E9	Action:T27 Acts-on:T28
T28	Reagent 539 567	reconstituted probe solution
T29	Temperature 571 587	room temperature
R11	Setting Arg1:E9 Arg2:T29	
T30	Action 589 592	Mix
E10	Action:T30 Using:T31
T31	Method 601 610	vortexing
T32	Action 617 627	centrifuge
E11	Action:T32 
T33	Action 691 694	add
E12	Action:T33 Acts-on:T38 Site:T40
T34	Action 640 647	prepare
E13	Action:T34 Creates:T35 Using:E12
T35	Reagent 652 672	Hybridization Buffer
T36	Reagent 684 689	probe
R12	Meronym Arg1:T36 Arg2:T35	
T37	Amount 695 699	1 μL
T38	Reagent 703 723	probe stock solution
T39	Amount 727 733	100 μL
T40	Reagent 737 757	Hybridization Buffer
R13	Measure Arg1:T40 Arg2:T39	
R14	Measure Arg1:T38 Arg2:T37	
T41	Action 768 774	vortex
E14	Action:T41 
T42	Action 779 789	centrifuge
E15	Action:T42 
T43	Amount 791 816	enough for one coverglass
T44	Action 824 831	creates
E16	Action:T44 Creates:T45
T45	Reagent 834 856	working probe solution
T46	Concentration 860 866	125 nM
R15	Measure Arg1:T45 Arg2:T46	
T47	Action 914 922	Aspirate
E17	Action:T47 Acts-on:T50 Site:T51
T48	Action 890 894	used
E18	Action:T48 Acts-on:T49
T49	Reagent 873 881	solution
T50	Reagent 927 936	MeOH-AcOH
T51	Location 945 955	coverglass
T52	Reagent 967 981	adherent cells
T53	Location 989 1006	the 12-well plate
R16	Meronym Arg1:T52 Arg2:T53	
R17	Meronym Arg1:T53 Arg2:T51	
T54	Action 1008 1011	Add
E19	Action:T54 Acts-on:T56
T55	Amount 1012 1016	1 mL
T56	Reagent 1020 1033	Wash Buffer A
T57	Action 1058 1066	incubate
E20	Action:T57 
T58	Temperature 1070 1086	room temperature
T59	Time 1091 1102	2-5 minutes
R18	Setting Arg1:E20 Arg2:T58	
R19	Setting Arg1:E20 Arg2:T59	
R20	Measure Arg1:T56 Arg2:T55	
T60	Action 1104 1112	Assemble
E21	Action:T60 Acts-on:T61
T61	Location 1113 1131	humidified chamber
T62	Size 1133 1139	150 mm
T63	Location 1140 1160	tissue culture plate
R21	Measure Arg1:T63 Arg2:T62	
T64	Action 1169 1174	lined
E22	Action:T64 Acts-on:T63 Using:T67 Using2:T66
T65	Modifier 1175 1181	evenly
T66	Device 1189 1221	flat water-saturated paper towel
T67	Device 1244 1252	Parafilm
T68	Amount 1226 1240	a single layer
R22	Measure Arg1:T67 Arg2:T68	
R23	Mod-Link Arg1:E22 Arg2:T65	
T69	Action 1253 1259	placed
E23	Action:T69 Site:T70 Acts-on:T67
T70	Location 1263 1266	top
T71	Mention 1274 1285	paper towel
R24	Coreference-Link Arg1:T71 Arg2:T66	
R25	Meronym Arg1:T70 Arg2:T71	
R26	Meronym Arg1:T63 Arg2:T61	
T72	Location 1390 1408	humidified chamber
T73	Action 1410 1418	dispense
E24	Action:T73 Acts-on:T76 Site:T77
T74	Amount 1419 1425	100 μL
T75	Reagent 1433 1453	Hybridization Buffer
T76	Reagent 1465 1470	probe
T77	Location 1480 1488	Parafilm
R28	Meronym Arg1:T75 Arg2:T76	
R27	Measure Arg1:T76 Arg2:T74	
R29	Meronym Arg1:T77 Arg2:T72	
T78	Amount 1548 1554	100 μL
T79	Reagent 1555 1559	drop
T80	Reagent 1563 1583	Hybridization Buffer
T81	Reagent 1595 1600	probe
R30	Measure Arg1:T79 Arg2:T78	
R31	Meronym Arg1:T81 Arg2:T79	
R32	Meronym Arg1:T80 Arg2:T81	
T82	Action 1497 1505	transfer
E25	Action:T82 Site:T79 Acts-on:T83
T83	Location 1510 1520	coverglass
T84	Modifier 1522 1537	cells side down
T85	Action 1602 1607	Cover
E26	Action:T85 Acts-on:T86 Using:T87
T86	Location 1612 1630	humidified chamber
T87	Seal 1640 1658	tissue culture lid
T88	Action 1664 1668	seal
E27	Action:T88 Using:T89 Acts-on:T86
T89	Device 1674 1682	Parafilm
T90	Action 1684 1692	Incubate
E28	Action:T90 
T91	Temperature 1708 1713	37 °C
T92	Time 1718 1725	2 hours
T93	Modifier 1693 1704	in the dark
R34	Mod-Link Arg1:E28 Arg2:T93	
R35	Setting Arg1:E28 Arg2:T91	
R36	Setting Arg1:E28 Arg2:T92	
T94	Time 1756 1769	up to 16 hour
T95	Action 1728 1738;1746 1755	Incubation continued
E29	Action:T95 
R37	Setting Arg1:E29 Arg2:T94	
T96	Action 1780 1788	transfer
E30	Action:T96 Acts-on:T97 Site:T99
T97	Location 1793 1803	coverglass
T98	Modifier 1805 1818	cells side up
T99	Location 1825 1844	fresh 12-well plate
T100	Amount 1856 1860	1 mL
T101	Reagent 1864 1877	Wash Buffer A
R38	Mod-Link Arg1:T97 Arg2:T98	
R33	Mod-Link Arg1:T83 Arg2:T84	
R39	Measure Arg1:T101 Arg2:T100	
R40	Meronym Arg1:T101 Arg2:T99	
T102	Action 1880 1888	Incubate
E31	Action:T102 
T103	Modifier 1889 1900	in the dark
T104	Temperature 1904 1909	37 °C
T105	Time 1914 1924	30 minutes
R41	Mod-Link Arg1:E31 Arg2:T103	
R42	Setting Arg1:E31 Arg2:T104	
R43	Setting Arg1:E31 Arg2:T105	
T106	Action 1926 1934	Aspirate
E32	Action:T106 Acts-on:T107
T107	Reagent 1935 1948	Wash Buffer A
T108	Action 1959 1962	add
E33	Action:T108 Acts-on:T110
T109	Amount 1963 1967	1 mL
T110	Reagent 1971 1989	DAPI nuclear stain
T111	Reagent 1991 2004	Wash Buffer A
T112	Concentration 2019 2026	5 ng/mL
T113	Reagent 2027 2031	DAPI
T114	Action 2036 2048	counterstain
E34	Action:T114 Acts-on:T115 Using:E33
T115	Reagent 2053 2059	nuclei
R44	Measure Arg1:T110 Arg2:T109	
R46	Measure Arg1:T113 Arg2:T112	
R45	Meronym Arg1:T113 Arg2:T111	
T116	Action 2061 2069	Incubate
E35	Action:T116 
T117	Modifier 2070 2081	in the dark
T118	Temperature 2085 2090	37 °C
T119	Time 2095 2105	30 minutes
R47	Mod-Link Arg1:E35 Arg2:T117	
R48	Setting Arg1:E35 Arg2:T118	
R49	Setting Arg1:E35 Arg2:T119	
T120	Action 2107 2115	Aspirate
E36	Action:T120 Acts-on:T121
T121	Reagent 2120 2140	DAPI staining buffer
T122	Action 2151 2154	add
E37	Action:T122 Acts-on:T124
T123	Amount 2155 2159	1 mL
T124	Reagent 2163 2176	Wash Buffer B
R50	Measure Arg1:T124 Arg2:T123	
T125	Action 2179 2187	Incubate
E38	Action:T125 
T126	Temperature 2191 2207	room temperature
T127	Time 2212 2223	2-5 minutes
R51	Setting Arg1:E38 Arg2:T126	
R52	Setting Arg1:E38 Arg2:T127	
T128	Action 2225 2228	Add
E39	Action:T128 Acts-on:T131 Site:T129
T130	Amount 2257 2262	15 μL
T131	Reagent 2267 2294	Vectashield Mounting Medium
R53	Measure Arg1:T131 Arg2:T130	
T129	Location 2302 2318	microscope slide
T132	Location 2330 2340	coverglass
T133	Action 2324 2329	mount
E40	Action:T133 Acts-on:T132 Site:T134
T134	Location 2350 2355	slide
T135	Modifier 2357 2372	cells side down
R54	Mod-Link Arg1:E40 Arg2:T135	
T136	Action 2381 2390	wick away
E41	Action:T136 Acts-on:T138 Site:T139
T137	Modifier 2374 2380	Gently
T138	Reagent 2398 2407	anti-fade
T139	Location 2417 2426	perimeter
T140	Location 2434 2444	coverglass
T141	Amount 2391 2397	excess
R55	Measure Arg1:T138 Arg2:T141	
R56	Mod-Link Arg1:E41 Arg2:T137	
R57	Meronym Arg1:T139 Arg2:T140	
T142	Action 2446 2450	Seal
E42	Action:T142 Acts-on:T143 Using:T144
T143	Location 2455 2475	coverglass perimeter
T144	Reagent 2481 2499	clear nail polish,
T145	Action 2504 2516	allow to dry
E43	Action:T145 Acts-on:T144
T146	Modifier 2532 2538	gently
T147	Action 2539 2548	wipe away
E44	Action:T147 Acts-on:T148 Site:T149 Using:T150
T148	Reagent 2549 2563	any dried salt
T149	Location 2572 2582	coverglass
T150	Reagent 2589 2594	water
R58	Mod-Link Arg1:E44 Arg2:T146