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Primary Cell Culture Sourcebook

Welcome! GIBCO 3 in vivo 3 GIBCO www.lifetechnologies.com/primarycells www.lifetechnologies.com/gibco 3D www.lifetechnologies.com/3d-cellculture www.lifetechnologies.com/transfection www.lifetechnologies.com/proteins

................................ 1...................... 1............................. 2..................... 2................................. 4............................... 4....................... 5................................. 6............................... 6....................... 7.............................. 8.............................. 8................... 9................................ 9................................ 9...................... 10................................. 11.................................. 11....................... 12................................ 13................................ 13...................... 13................................ 14................................ 14...................... 14................................. 15.................................... 16............................... 20 3D................................ 23................................ 25 Neon Transfection System.......................... 26..................... 26................................ 27

(BPE) FREE A N O R I M A L I G I N (HIV-1 B C ) 70% 500,000cells (HEKn) HKGS S7 EpiLife Medium 30(HEKa) HKGS S7 EpiLife Medium25 BPE BPE HEKn-APFHEKa-APF EpiLife Medium S7 / Synth-a-Freeze HEKnHEKa EpiLife Medium EDGS /EDTA / Synth-a-Freeze HEKnHEKa EpiLife Medium HKGS /EDTA / Synth-a-Freeze HEKnHEKa Medium 154 HKGS /EDTA / Synth-a-Freeze 1

(HEK) A N O R I M A L FREE I G I N HEKn-APF ( R-011-K 15) EpiLife A N O R I M A L FREE I G I N (15 ) EpiLife in vitro 60 M 1 ( >500,000cells) C-020-5C M-EPI-500-CA HEKn 1 ( >500,000cells) C-001-5C (HEK) HEKa-APF ( R-011-K 15) 1 ( >500,000cells) C-021-5C A N O I M A L FREE R I G I N FREE EpiLife CF() (3 ) M-EPIcf-500 Medium 154CF Medium 154CF/PRF 0.5 M EpiLife CF/PRF( FREE ) EpiLife CF/PRF EpiLife (3 ) A N O R I M A L I G I N A N O R I M A L I G I N M-EPIcf/PRF-500 HEKa 1 ( >500,000cells) C-005-5C Medium 154 HKGS( S-001-5) HKGS( S-001-K) 200 M 2 M-154-500

Medium 154CF() Medium 154CF Medium 154 HKGS( S-001-5)HKGS( S-001-K) M-154CF-500 Medium 154CF/PRF( ) Medium 154CF/PRF Medium 154 HKGS( S-001-5) HKGS( S-001-K) M-154CF/PRF-500 EpiLife CF CF/PRF 0.65 M (HKGS) EpiLife Medium Medium 154 (100 ) (BPE)* 5ml S-001-5 EpiLife MediumMedium 154 *BPE (HKGS) EpiLife Medium Medium 154HKGS HKGS (BPE)* / B (GA) GA 1 S-001-K EpiLife MediumMedium 154 EpiLife +HKGS EpiLife (EDGS) EpiLife Medium (100 ) (Medium 154 ) BSA( >95% Medium 154+HKGS HEKn 1 HEKn 3 HEKn 5 EpiLife Medium 154HEKn( C-001-5C) <0.005%FA) ( >95%) rhigf-1( >95%) rhegf( >95%) ( >96%) PGE-2( >98%) EDGS ( R-011-K 15) 5ml S-012-5 3

FREE S7 EpiLife Medium (100 ) S7 S7 5ml EpiLife Medium 154 S7 ( R-011-K 15) A N O R I M A L 5ml EpiLife Medium I G I N S-017-5 10 60mM(10 0.5ml) S-013-EPI-10 200mM(10 0.5ml) S-013-154-10 (15) A N O R I M A L FREE I G I N (HIV-1 B C ) 70% 500,000cells HEMn-MP HEMn-DP12 HEMn-LP HEMa-LP16 4 HEMa-LP Mel-5 (HEM) HEMn-LP 1 ( >500,000cells) C-002-5C HEMn-MP 1 ( >500,000cells) C-102-5C HEMn-DP 1 ( >500,000cells) C-202-5C (HEM) HEMa-LP -2( S-016-5) 1 ( >500,000cells) C-024-5C 4

Medium 254 HMGS( S-002-5)HMGS-2( S-016-5) M-254-500 Medium 254CF() Medium 254CF * Medium 254 HMGS( S-002-5)HMGS-2( S-016-5) M-254CF-500 * Medium 254CF 0.5 M (HMGS) Medium 254 Medium 254CF (100 ) (BPE) 13-12- 5ml S-002-5 BPE -2(HMGS-2) Medium 254 Medium 254CF (100 ) (BPE) -1 HEMa-LP 5ml S-016-5 (15) BPE HEMa 6 Mel-5 HEMa 6 5

(HIV-1 B C ) 70% 500,000cells HDFa12 HDFn 16 (HDF) HDFn 1 ( >500,000cells) C-004-5C (HDF) HDFa 1 ( >500,000cells) C-013-5C 6

Medium 106 LSGS( S-003-10)LSGS( S-003-K) M-106-500 (LSGS) LSGS LSGS / (BSA ) / B (GA) GA 1 S-003-K (15) (LSGS) Medium 106 (50 ) 10ml S-003-10 HDFn 3 HDFn 5 HDFa 5 HDFa 8 15-27 3D 7

(HIV-1 B C ) 70% 500,000cells 16 dii-ac-ldl von Willebrand (vwf) CD31CD36( ) - ( ) dii-ac-ldl -vwf -CD36 -CD31 (HMVEC) HMVECnd HMVECad 1 HMVECad 3 1 ( >500,000cells) C-010-5C (HMVEC) HMVECad HMVECad 5 1 ( >500,000cells) C-011-5C 8

Medium 131+ MVGS( S-005-25) 1 (100ml S-006-100) (MVGS) Medium 131 (20 ) dbcamp M-131-500 25ml S-005-25 (15) (AF) (1X) AF (HIV-1 B C ) 70% 16 dii-ac-ldl von Willebrand (vwf) CD31( ) - ( ) 100ml S-006-100 (15) HAEC 1 ( >500,000cells) C-006-5C HPAEC 1 ( >500,000cells) C-008-5C 9

HUVEC 1 ( >500,000cells) C-003-5C HUVECp Medium 200 LSGS( S-003-10)LSGS( S-003-K) M-200-500 >500,000 / 1 C-015-5C ( >500,000cells) >1,000,000 / 1 C-015-10C ( >1,000,000cells) Medium 200PRF() Medium 200PRFMedium 200 LSGS( S-003-10) LSGS( S-003-K) M-200PRF-500 HUVEC 3 HUVEC 5 HUVEC 7 HUVEC -CD31 HUVEC dii-ac-ldl HUVEC -vwf 10

11 (LSGS) Medium 200 Medium 200PRF (50 ) 10ml S-003-10 (LSGS) LSGS LSGS / (BSA ) / B (GA) GA 1 S-003-K (15) (HIV-1 B C ) 70% 500,000cells16 SMDS- ( )von Willebrand (vwf ) HASMC 1 ( >500,000cells) C-007-5C HCASMC 1 ( >500,000cells) C-017-5C HPASMC 1 ( >500,000cells) C-009-5C 3D 15-27 HASMC 5 HPASMC 5 HCASMC 5

Medium 231 SMGS( S-007-25) SMDS( S-008-5) M-231-500 / (SMGS) Medium 231 (20 ) BSA 25ml S-007-25 (SMDS) Medium 231 (100 ) 5ml S-008-5 (15) 12

13 EpiLife Medium HCGS( S-009-5) 60 M M-EPI-500-CA EpiLife CF() * HCGS( S-009-5) M-EPIcf-500 HCEC-2 5 HCEC-2 9 HCEC-2 7 HCEC-3 5 HCEC-3 9 HCEC-3 7 (HIV-1 B C ) 70% 500,000cells (HCEC) HCEC-2 1 ( >500,000cells) C-018-5C EpiLife CF/PRF( ) EpiLife CF/PRF * EpiLife HCGS( S-009-5) M-EPIcf/PRF-500 * EpiLife CFEpiLife CF/PRF 0.65 M (HCGS) EpiLife (100 ) (BPE) () 5ml S-009-5 (15) BPE

5/6 8 18E- 26 HMEC 1 ( >500,000cells) A10565 (MEGS) Medium 171 (100 ) (BPE)* 5ml S-015-5 *BPE Medium 171 MEGS( S-015-5) M-171-500 30 25 20 MEGM M171/MEGS 106 106 15 10 5 0 0 5 10 15 20 25 30 35 126 126 Medium 171/MEGS2 (MEGS) Medium 171 (HMEC) HMEC (8 )9 14

/EDTA (TE) EDTA (1X) 100ml R-001-100 Synth-a-Freeze 10% DMSO 50ml A12542-01 A N O I M A L FREE R I G I N (TN) (1X) 10 A N O I M A L FREE R I G I N 100ml R-002-100 60mM 10 0.5ml S-013-EPI-10 200mM 10 0.5ml S-013-154-10 (DTI) (1X) 100ml A N O R I M A L FREE I G I N R-007-100 / B FREE (GA) 10 A N O R I M A L I G I N B (500X) (10 1ml / ) GA 10 10 10 1ml R-015-10 30 T-25 1 R-011-K A N O R I M A L FREE I G I N 15

www.lifetechnologies.com/gibco Clostridium histolyticum (Clostridiopeptidase A) E. C. 3.4.2.4.3 125 /mg37 181 L- 1 mol Bacillus polymyxa 5g 17105-041 500mg 17018-029 Fungizone TM 1ml250 g B Clostridium histolyticum 1g 17100-017 0.25-2.5 g/ml Fungizone TM E.R. Squibb & Sons, L.L.C. 20ml 15290-018 Clostridium histolyticum 1g 17101-015 Clostridium histolyticum 50mg/ml 0.5-50 g/ml 10mg/ml 10ml 15710-064 10 10ml() 15710-072 50mg/ml 10ml 15750-060 10 10ml() 15750-078 1g 17104-019 16

Recovery TM Recovery TM Recovery TM 25% - -DMSO DulbeccoEagle Medium(DMEM)10% DMSO 50ml 12648-010 Recovery TM Medium 110 100 90 80 70 60 50 40 30 20 10 0 AE-1 CHO MDCK Swiss 3T3 HeLa Recovery TM CCFM12648-010 S S SC C SM GM BL 8 5 12-2.3 10 4 /6.0 10 5 / (AE-1 2 CHO 6 MDCK 6 Swiss 3T3 5 HeLa 6 ) Recovery TM (CCFM)(n=4) TrypLE TM Select(1X) 1mM EDTAD-PBS 15-30 6 100ml 12563-011 12563-029 17

TrypLE TM Express(1X) 0.6 TrypLE TM Express TrypLE TM Select AU 0.5 0.4 0.3 1mM EDTAD-PBS 0.2 0.1 15-30 6 0.0 1 3 5 7 9 11 13 15 17 19 100ml 12605-010 12605-028 20 100ml() 12605-036 1. 2.4 TrypLE TM Express() (1X) 1mM EDTAD-PBS AU 2.0 1.6 1.2 0.8 TrypLE 0.4 100ml 12604-013 12604-021 20 100ml() 12604-039 0.0 1 3 5 7 9 11 13 15 2. TrypLE TM 0.25%(1X) Hank2.5g/L (1:250) (MgCl 2 6H 2 O) (MgSO 4 7H 2 O) 140 120 100 80 60 40 20 TrypLE 100ml 15050-065 15050-057 0 3. TrypLE TM TrypLE TM Express TrypLE TM Express 1 HPLC 2 TrypLE TM Express 18

2.5%(10X) 25g/L(1:250) 8.5g/L 1X 2.5g(1:250)/L -EDTA (0.5%EDTA 4Na)(10X) 5.0g/L(1:250) 2.0g/L EDTA 4Na 8.5g/L 1X 0.5g0.2g EDTA 4Na/L(1:250) 100ml 15090-046 100ml 15400-054 -EDTA (0.05%EDTA 4Na)(1X) Hank 0.5g/L(1:250) 0.2g/L EDTA 4Na (MgCl 2 6H 2 O) (MgSO 4 7H 2 O) -EDTA (0.25%EDTA 4Na)(1X) Hank 2.5g/L(1:250) 0.38g/L EDTA 4Na (MgCl 2 6H 2 O) (MgSO 4 7H 2 O) 100ml 25300-054 20 100ml() 25300-120 25300-062 100ml 25200-056 20 100ml() 25200-114 25200-072 19

GIBCO (SFM) Serum-free media-gibco Protein-free media-gibco Chemically difined media-gibco Animal origin-free media-gibco ( ) Neurobasal Medium Neurobasal Medium 21103-049 12348-017 Neurobasal -A Medium 10888-022 Neurobasal -A Medium (1 ) 12349-015 B-27 AO 10ml 17504-044 B-27 AO B-27 (AOB-27 10ml 10889-038 B-27 A( ) ) CNS 10ml 12587-010 N-2 G-5 (PC12, B104, N1E- 115NS20) (U-251, MGsp, C62BD, RN-22) ( <125 g/ ml) 5ml 17502-048 1ml 17503-012 20

AIM V Medium* HSA AIM V Medium IL-2 ex vivo (TIL ) T (GM-CSF ) StemPro -34 (StemPro ) * (CD34+) ( )/ 1,000ml 1,000ml 10L 12055-091 12055-083 087-0112DK 087-0112BK 12065-074 10639-011 BG01V/hOG hesc hoct4-gfp StemPro hesc StemPro EZPassage KnockOut (KSR) (GFP) Oct-4 BG01V feeder (ESC) FBS hescs ( ) BG01 BG02 BG03 H1 H9 BG01V HUES6 HUES9 CyT49 Cyth203 ES (R366.4) ESCESC / ESC in vitro KSR FBS KnockOut DMEM KnockOut DMEM/F-12 ES ( ) ES (USDA ) StemPro MesenPRO RS Medium MSC (USDA ) ADSC- 1 (2%) ADSC 1 ~2 10 6 R7799-105 * 1 A10007-01 1 10 23181-010 10828-028 10829-018 12660-012 100ml 100ml 16141-061 16141-079 10439-016 10439-024 1 R7788-110 1 12746-012 100ml 12662-011 12662-029 21

bfgf EGF ( ) 11111-044 - (EGF BPE )* ( )* CD ( ) ( ) (9 P450) in vitro BPE (<25 g/ml) 17705-021 17005-042 37010-022 10744-019 HuMEC 12753-108 HuMEC 12753-010 HuMEC 1 12755-013 5ml( )25mg KnockOut D-MEM (ES) ES 10829-018 KnockOut ES 10828-028 LHC 1,000ml 12677-019 12677-027 LHC-8 12678-017 LHC-8 ( ) 12679-015 LHC-9 12680-013 CD- 1ml 12332-011 * 22

3D in vivo in vivo Geltrex TM (LDEV ) Geltrex TM (RGF) (BME) Engelbreth-Holm-Swarm(EHS) entactin (3D) Geltrex TM Geltrex TM Geltrex TM LDEV(Lactete Dehydrogenase-Elevating Virus: ) in vivo 3D 3D 10 g/ml DulbeccoEagle LDEV SVEC4-105%FBS Geltrex TM 37 24 Geltrex TM Geltrex TM 1 ml A1413201 12760-013* 5 ml A1413202 12760-021* 1 ml A1413301 A1048001* 5 ml A1413302 A1048002* *2012 3 19 LDEV 23

GIBCO AlgiMatrix TM 3D AlgiMatrix TM 3D 3 500 MCTS 3 / 10 6 / 400 300 200 100 2D 2% AlgiMatrix TM Algimatrix TM Firming Buffer ( A1091501) 6wells Kit 3 1 0 3-MC 3-MC 3-MC 3-MC Furafylline 24 Furafylline 48 1 C3A CYP1A2( P450)3-MC() CYP1A2 2 A B AlgiMatrix TM 3D(MCTS) 1 2-4 5 6 1 3D 7 in vitro C D 1. Kunz-Schughart, L., et al., (2004) J Biomolec Screening 9, 273. 2. Dvir-Ginzberg, M., et al. (2003) Tissue Engineering 9, 757. 3. Elkayam, T., et al. (2006) Tissue Engineering 12, 1357. 4. Kedem, A., et al. (2005) Tissue Engineering 11, 715. 5. Dar, A., et al. (2002) Biotechnology & Bioengineering 80, 305. 6. Kim, J. (2005) Seminars in Cancer Biology 15, 365. 7. Gerecht-Nir, S., et al. (2004) Biotechnology & Bioengineering 88, 313. 2 AlgiMatrix TM A. B. 8- ( )propidium iodide C. ( ) D. PCNA ( ) AlgiMatrix TM 3D Culture System in 96-wells plate 96 /1 12684-015 AlgiMatrix TM 3D Culture System in 24-wells plate 24 /1 12684-023 AlgiMatrix TM 3D Culture System in 96-wells plate 96 /5 12684-031 AlgiMatrix TM 3D Culture System in 24-wells plate 24 /4 12684-049 AlgiMatrix TM 3D Firming Buffer 50 ml A1091501 AlgiMatrix TM 3D Culture System in 6-wells plate and Buffer 1kit (6 /1) A1098201 AlgiMatrix TM 3D Culture System in 6-wells plate and Buffer 1kit (6 /4) A1098202 24

DNA RNA BT Lipofectamine TM LTX PLUS TM Lipofectamine TM LTX 90% Lipofectamine TM LTXPLUS TM (HUVEC) HUVEC (Matrigel) 1 2 Lipofectamine TM LTX HUVEC (>50%) Matrigel Lipofectamine TM LTXPLUS TM 1. Bernardini et al. (2000) I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo. Blood 96, 4039 4045. 2. Mukherjee et al. (2005) Cytotoxic and anti-angiogenic activity of AW464 (NSC 706704), a novel thioredoxin inhibitor: An in vitro study. British Journal of Cancer 92, 350 358. www.lifetechnologies.com/transfection Lipofectamine LTX 1.0ml 15338-100 PLUS 0.85ml 11514-015 0ng 3D 5% 10% Lipofectamine TM LTXPLUS TM 50% 500ng 1,000ng Lipofectamine TM LTXPLUS TM PLUS TM (2.5 l) Lipofectamine TM LTX (1.5 l) GFP- 24 GFP Beverly Davidson 25

Neon Transfection System DNAsiRNA 80% Neon Transfection Neon Transfection 24 www.lifetechnologies.com/neon Protocol Exchange www.protocolexchange.com - - -GIBCO Neon Transfection Neon Transfection System 1 1 MPK5000 Neon Transfection System Starter Pack 2 1 MPK5000S Neon Transfection System Kit (100 µl) 1 MPK10096 Neon Transfection System Kit (10 µl) 1 MPK1096 Neon Transfection Tubes (100 ) 1 MPT100 Neon Pipette MPP100 Neon Pipette Station MPS100 1 Neon Transfection System(MPK5000) Neon Neon Pipette(MPP100) Neon Pipette Station(MPS100) 2 Starter Pack(MPK5000S)Neon Transfection System (MPK 5000) Transfection Kit(MPK10096 MPK1096 1 ) www.lifetechnologies.com/proteins OD 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 SCFM07e ED 50 (pg/ml) BioSource = 548.4 = 6,593.6 0.4 10 0 10 1 10 2 10 3 10 4 10 5 10 6 SCF (pg/ml) (SCF) M07e SCF SCF (ED 50 ) 26

Human proteins Recombinant human Activin A 5 µg PHG9014 Recombinant human BDNF 10 µg PHC7074 Recombinant human BMP-4 10 µg PHC7914 Recombinant human EGF 200 µg PHG0064 Recombinant human FGFb (truncated) 10 µg PHG0024 Recombinant human FGFb (full length) 10 µg PHG0264 Recombinant human Flt-3 ligand 10 µg PHC1704 Recombinant human EGF 100 µg PHG0311 Recombinant human GDNF 10 µg PHC7045 Recombinant human GM-CSF 10 µg PHC2015 Recombinant human HGF 10 µg PHG0254 Recombinant human IFN-gamma 100 µg PHC4031 Recombinant human IGF-1 200 µg PHG0075 Recombinant human IL-1 beta 10 µg PHC0815 Recombinant human IL-2 10 µg PHC0026 Recombinant human IL-3 10 µg PHC0034 Recombinant human IL-4 10 µg PHC0044 Recombinant human IL-6 10 µg PHC0065 Recombinant human IL-7 25 µg PHC0076 Recombinant human IL-8 25 µg PHC0084 Recombinant human IL-21 10 µg PHC0214 Recombinant human IL-33 10 µg PHC0334 Recombinant human M-CSF 10 µg PHC2044 Recombinant human Noggin 20 µg PHC1506 Recombinant human PDGF BB 10 µg PHG0045 Recombinant human SCF/cKit 10 µg PHC2115 Recombinant human SDF-1 a 10 µg PHC1344 Recombinant human TGF-beta 1 5 µg PHG9204 Recombinant human TNF-alpha 50 µg PHC3016 Mouse proteins Recombinant mouse Fgf basic 10 µg PMG0034 Recombinant mouse G-CSF 10 µg PMC2034 Recombinant mouse GM-CSF 10 µg PMC2015 Recombinant mouse IFN alpha 10 5 units PMC4016 Recombinant mouse IFN Beta 10 5 units PMC4024 Recombinant mouse IFN gamma 15 µg PMC4034 Recombinant mouse IL-3 10 µg PMC0034 Recombinant mouse IL-4 10 µg PMC0045 Recombinant mouse IL-6 10 µg PMC0065 Recombinant mouse IL-12 10 µg PMC0126 Recombinant mouse IL-33 10 µg PMC0334 Mouse Laminin (natural) 1 mg 23017015 Recombinant mouse PDGF BB 10 µg PMG0044 Recombinant mouse SCF/cKit 10 µg PMC2114 Recombinant mouse Sonic Hedgehog 10 µg PMC2094 Recombinant mouse TNF alpha 10 µg PMC3014 www.lifetechnologies.com/proteins 27

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