protocol_351.ann

T1	Action 66 70	Warm
E1	Action:T1 Acts-on:T50
T2	Action 164 171	aliquot
E2	Action:T2 Acts-on:T55 Site:T57
T3	Action 240 244	warm
E3	Action:T3 Acts-on:T57
T4	Action 254 259	Chill
E4	Action:T4 Acts-on:T59
T5	Action 325 332	aliquot
E5	Action:T5 Acts-on:T64
T6	Action 370 375	chill
E6	Action:T6 
T7	Action 386 393	Aliquot
E7	Action:T7 Acts-on:T67 Site:T69
T8	Action 612 619	Prepare
E8	Action:T8 Acts-on:T71 Count:T70
T9	Action 704 712	Incubate
E9	Action:T9 Acts-on:T72 Using:T73
T10	Action 791 794	Fix
E10	Action:T10 Acts-on:T76 Using:T80
T11	Action 887 894	pipette
E11	Action:T11 
T12	Action 923 931	Incubate
E12	Action:T12 
T13	Action 992 1002	Centrifuge
E13	Action:T13 Acts-on:T88
T14	Action 1055 1061	decant
E14	Action:T14 Acts-on:T92
T15	Action 1075 1081	vortex
E15	Action:T15 
T16	Action 1085 1094	resuspend
E16	Action:T16 Acts-on:T93 Using:E15
T17	Action 1109 1112	Add
E17	Action:T17 Acts-on:T95
T18	Action 1148 1152	wash
E18	Action:T18 Acts-on:T96
T19	Action 1264 1274	centrifuge
E19	Action:T19 
T20	Action 1325 1331	decant
E20	Action:T20 Acts-on:T101
T21	Action 1345 1351	Repeat
E21	Action:T21 
T22	Action 1388 1395	pipette
E22	Action:T22 Acts-on:T102
T23	Action 1427 1436	resuspend
E23	Action:T23 Using:T105 Acts-on:T104
T24	Action 1468 1480	permeabilize
E24	Action:T24 Acts-on:T106 Using:E25
T25	Action 1490 1496	adding
E25	Action:T25 Acts-on:T108
T27	Action 1632 1640	Incubate
E27	Action:T27 
T28	Action 1710 1720	Centrifuge
E28	Action:T28 Acts-on:T112
T29	Action 1774 1780	decant
E29	Action:T29 Acts-on:T116
T30	Action 1794 1800	vortex
E30	Action:T30 
T31	Action 1804 1813	resuspend
E31	Action:T31 Using:E30 Acts-on:T117
T32	Action 1828 1831	Add
E32	Action:T32 Acts-on:T119
T33	Action 1867 1871	wash
E33	Action:T33 Acts-on:T120 Using:E32
T34	Action 1883 1893	centrifuge
E34	Action:T34 Acts-on:T121
T35	Action 1947 1953	decant
E35	Action:T35 Acts-on:T125
T36	Action 2003 2012	Resuspend
E36	Action:T36 Acts-on:T126 Site:T128
T38	Action 2213 2221	Transfer
E38	Action:T38 Acts-on:T37 Site:T131
T39	Action 2252 2255	Add
E39	Action:T39 Acts-on:T132 Site:T133
T40	Action 2299 2305	vortex
E40	Action:T40 Acts-on:T133
T41	Action 2318 2326	incubate
E41	Action:T41 Acts-on:T136
T42	Action 2309 2312	mix
E42	Action:T42 Using:E40
T43	Action 2376 2379	Add
E43	Action:T43 Acts-on:T138
T44	Action 2410 2420	centrifuge
E44	Action:T44 Acts-on:T139
T45	Action 2474 2480	decant
E45	Action:T45 Acts-on:T143
T46	Action 2494 2500	Repeat
E46	Action:T46 
T47	Action 2530 2539	Resuspend
E47	Action:T47 Acts-on:T144 Site:T146
T48	Action 2598 2605	analyze
E48	Action:T48 Using:T147 Acts-on:T144
T49	Concentration 71 74	1 X
T50	Reagent 75 102	RBC Lysis/Fixation Solution
T51	Amount 141 147	0.1 mL
T52	Reagent 151 162	whole blood
T53	Amount 172 176	2 mL
T54	Concentration 180 183	1 X
T55	Reagent 184 211	RBC Lysis/Fixation Solution
T56	Size 217 222	50 mL
T57	Location 223 235	conical tube
T58	Temperature 248 253	37°C.
T59	Reagent 260 282	True-Phos™ Perm Buffer
T60	Temperature 286 291	-20°C
T61	Amount 302 308	0.1 mL
T62	Reagent 312 323	whole blood
T63	Amount 333 339	1.0 mL
T64	Reagent 343 365	True-Phos™ Perm Buffer
T65	Temperature 379 384	-20°C
T66	Amount 394 400	0.1 mL
T67	Reagent 404 415	whole blood
T68	Size 433 438	50 mL
T69	Location 439 451	conical tube
T70	Numerical 620 623	two
T71	Reagent 624 632	aliquots
T72	Reagent 717 722	cells
T73	Reagent 732 751	appropriate stimuli
T74	Temperature 760 780	suitable temperature
T75	Time 785 789	time
T76	Reagent 799 804	cells
T77	Modifier 805 816	immediately
T78	Modifier 836 846	pre-warmed
T79	Concentration 847 850	1 X
T80	Reagent 851 878	RBC Lysis/Fixation Solution
T81	Action 914 920	mixing
E49	Action:T81 Using:E11
T82	Modifier 905 913	thorough
T83	Modifier 880 886	Gently
T84	Temperature 935 939	37°C
T85	Time 944 954	15 minutes
T86	Action 984 989	fixed
E50	Action:T86 Acts-on:T87
T87	Reagent 965 970	cells
T88	Reagent 1003 1008	cells
T89	Speed 1012 1019	350 x g
T90	Temperature 1023 1039	room temperature
T91	Time 1044 1053	5 minutes
T92	Reagent 1062 1073	supernatant
T93	Reagent 1095 1106	cell pellet
T94	Amount 1113 1123	sufficient
T95	Reagent 1124 1144	Cell Staining Buffer
T96	Reagent 1157 1162	cells
T97	Reagent 1207 1237	BioLegend Cell Staining Buffer
T98	Speed 1278 1285	350 x g
T99	Temperature 1289 1305	room temperature
T100	Time 1310 1319	5 minutes
T101	Reagent 1332 1343	supernatant
T102	Reagent 1396 1401	cells
T103	Modifier 1381 1387	Gently
T104	Reagent 1437 1448	cell pellet
T105	Reagent 1408 1423	residual volume
T106	Reagent 1481 1486	cells
T107	Modifier 1497 1508	pre-chilled
T108	Reagent 1509 1531	True-Phos™ Perm Buffer
T26	Temperature 1644 1649	-20°C
T109	Time 1654 1664	60 minutes
T110	Action 1694 1707	permeabilized
E26	Action:T110 Acts-on:T111 Using:E27
T111	Reagent 1675 1680	cells
T112	Reagent 1721 1726	cells
T113	Speed 1730 1738	1000 x g
T114	Temperature 1742 1758	room temperature
T115	Time 1763 1772	5 minutes
T116	Reagent 1781 1792	supernatant
T117	Reagent 1814 1825	cell pellet
T118	Amount 1832 1842	sufficient
T119	Reagent 1843 1863	Cell Staining Buffer
T120	Reagent 1876 1881	cells
T121	Reagent 1894 1899	cells
T122	Speed 1903 1911	1000 x g
T123	Temperature 1915 1931	room temperature
T124	Time 1936 1945	5 minutes
T125	Reagent 1954 1965	supernatant
T126	Reagent 2017 2022	cells
T127	Action 1967 1973	Repeat
E51	Action:T127 
T128	Reagent 2038 2058	Cell Staining Buffer
T129	Amount 2077 2101	starting volume of blood
T37	Reagent 2222 2228	100 ml
T130	Size 2234 2244	12 x 75 mm
T131	Location 2245 2249	tube
T132	Reagent 2256 2276	antibody cocktail(s)
T133	Location 2280 2297	appropriate tubes
T134	Time 2331 2341	30 minutes
T135	Temperature 2345 2361	room temperature
T137	Amount 2380 2384	2 mL
T136	Location 2369 2373	dark
T138	Reagent 2388 2408	Cell Staining Buffer
T139	Reagent 2421 2426	cells
T140	Speed 2430 2438	1000 x g
T141	Temperature 2442 2458	room temperature
T142	Time 2463 2472	5 minutes
T143	Reagent 2481 2492	supernatant
T144	Reagent 2540 2545	cells
T145	Amount 2563 2569	500 uL
T146	Reagent 2573 2593	Cell Staining Buffer
T147	Device 2613 2627	flow cytometer
R1	Measure Arg1:T50 Arg2:T49	
R2	Measure Arg1:T52 Arg2:T51	
R3	Measure Arg1:T55 Arg2:T54	
R4	Measure Arg1:T55 Arg2:T53	
R5	Meronym Arg1:T55 Arg2:T52	
R6	Measure Arg1:T57 Arg2:T56	
R7	Setting Arg1:E3 Arg2:T58	
R8	Setting Arg1:E4 Arg2:T60	
R9	Measure Arg1:T64 Arg2:T63	
R10	Meronym Arg1:T64 Arg2:T62	
R11	Measure Arg1:T62 Arg2:T61	
R12	Setting Arg1:E6 Arg2:T65	
R13	Measure Arg1:T67 Arg2:T66	
R14	Measure Arg1:T69 Arg2:T68	
R15	Setting Arg1:E9 Arg2:T74	
R16	Setting Arg1:E9 Arg2:T75	
R17	Mod-Link Arg1:E10 Arg2:T77	
R18	Mod-Link Arg1:T80 Arg2:T78	
R19	Measure Arg1:T80 Arg2:T79	
R20	Mod-Link Arg1:E11 Arg2:T83	
R21	Mod-Link Arg1:E49 Arg2:T82	
R22	Setting Arg1:E12 Arg2:T84	
R23	Setting Arg1:E12 Arg2:T85	
R24	Setting Arg1:E13 Arg2:T89	
R25	Setting Arg1:E13 Arg2:T90	
R26	Setting Arg1:E13 Arg2:T91	
R27	Measure Arg1:T95 Arg2:T94	
R28	Or Arg1:T95 Arg2:T97	
R29	Setting Arg1:E19 Arg2:T98	
R30	Setting Arg1:E19 Arg2:T99	
R31	Setting Arg1:E19 Arg2:T100	
R32	Mod-Link Arg1:E22 Arg2:T103	
R33	Mod-Link Arg1:T108 Arg2:T107	
R34	Setting Arg1:E27 Arg2:T26	
R35	Setting Arg1:E27 Arg2:T109	
R36	Setting Arg1:E28 Arg2:T113	
R37	Setting Arg1:E28 Arg2:T114	
R38	Setting Arg1:E28 Arg2:T115	
R39	Measure Arg1:T119 Arg2:T118	
R40	Setting Arg1:E34 Arg2:T122	
R41	Setting Arg1:E34 Arg2:T123	
R42	Setting Arg1:E34 Arg2:T124	
R43	Measure Arg1:T128 Arg2:T129	
R44	Measure Arg1:T131 Arg2:T130	
R45	Setting Arg1:E41 Arg2:T134	
R46	Setting Arg1:E41 Arg2:T135	
R47	Measure Arg1:T138 Arg2:T137	
R48	Setting Arg1:E44 Arg2:T140	
R49	Setting Arg1:E44 Arg2:T141	
R50	Setting Arg1:E44 Arg2:T142	
R51	Measure Arg1:T146 Arg2:T145