Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet:

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific Gibco™ 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific Gibco™ 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific Gibco™ 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific Gibco™ 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific Gibco™ 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Concentration Assay

Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet: Cells were fixed with 4% paraformaldehyde 14 days after isolation and subsequently stained for CD31 (BD, 1:500) as an endothelial cell specific marker in combination with α-SMA (pericytes and smooth muscle cells, Acris, 1:200, upper row), Iba1 (microglia, Wako Pure Chemical Industries, 1:100, middle row) and GFAP (astrocytes, Millipore, 1:400, lower row). Scale bars represent 50 μm.

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific Gibco™ 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific Gibco™ 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific Gibco™ 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific Gibco™ 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific Gibco™ 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Isolation, Staining, Marker

Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet:

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific Gibco™ 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific Gibco™ 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific Gibco™ 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific Gibco™ 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific Gibco™ 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Concentration Assay

Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet:

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific GibcoTM 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific GibcoTM 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific GibcoTM 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific GibcoTM 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific GibcoTM 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Concentration Assay, Sterility, Clinical Proteomics

Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet:

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific GibcoTM 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific GibcoTM 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific GibcoTM 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific GibcoTM 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific GibcoTM 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Concentration Assay, Sterility, Clinical Proteomics

Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet:

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific GibcoTM 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific GibcoTM 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific GibcoTM 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific GibcoTM 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific GibcoTM 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Concentration Assay, Sterility, Clinical Proteomics

Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet:

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific GibcoTM 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific GibcoTM 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific GibcoTM 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific GibcoTM 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific GibcoTM 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Concentration Assay, Sterility, Clinical Proteomics

Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet:

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific GibcoTM 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific GibcoTM 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific GibcoTM 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific GibcoTM 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific GibcoTM 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Concentration Assay, Sterility, Clinical Proteomics

Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet: Cells were fixed with 4% paraformaldehyde 14 days after isolation and subsequently stained for CD31 (BD, 1:500) as an endothelial cell specific marker in combination with α-SMA (pericytes and smooth muscle cells, Acris, 1:200, upper row), Iba1 (microglia, Wako Pure Chemical Industries, 1:100, middle row) and GFAP (astrocytes, Millipore, 1:400, lower row). Scale bars represent 50 μm.

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific GibcoTM 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific GibcoTM 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific GibcoTM 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific GibcoTM 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific GibcoTM 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Isolation, Staining, Marker

Journal: Bio-protocol

Article Title: Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

doi: 10.21769/BioProtoc.2294

Figure Lengend Snippet:

Article Snippet: Geyer 2270 RT 4 Isoflurane Baxter KDG9623 RT 5 Collagenase/dispase (sterile filtered) Roche Diagnostics 11097113001 500 mg/5 ml H 2 O, sterile 200 μl -20 °C 100 mg/ml 1 mg/ml 100 μl collagenase/dispase in 10 ml medium 6 DNase I Roche Diagnostics 11284932001 100 mg/10 ml H 2 O, sterile 100 μl -20 °C 10 mg/ml 4 μg/ml 40 μl DNase I in 10 ml medium 7 N α -Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) Sigma-Aldrich 90182 14.7 mg/10 ml H 2 O, sterile 200 μl -20 °C 1.47 mg/ml diluted to 14.7 μg/ml 0.147 μg/ml 100 μl TLCK in 10 ml medium 8 Puromycin Sigma-Aldrich P8833 2.5 mg/10 ml H 2 O, sterile 500 μl -20 °C 0.25 mg/ml 8 μg/ml 32 μl puromycin in 1 ml medium 9 Trypsin-EDTA 0.25% Thermo Fisher Scientific GibcoTM 25200-056 100 ml -20 °C 1x 1x undiluted 10 4% paraformaldehyde Merck Millipore 1040051000 4% paraformaldehyde in DPBS -20 °C 11 CD31 BD BD Pharmingen 557355 1:500 12 α-smooth muscle Actin (α-SMA) Acris Antibodies DM001-05 1:200 13 Iba1 Wako Pure Chemical Industries 019-19741 1:100 14 Glial Fibrillary Acidic Protein (GFAP) EMD Millipore AB5541 1:400 15 Zona occludens-1 (ZO-1) Thermo Fisher Scientific Invitrogen 40-2200 1:500 16 VE-Cadherin Santa Cruz Biotechnology sc-6458 1:500 17 Claudin-5 (Cldn-5) Thermo Fisher Scientific Invitrogen 34-1600 1:500 18 Occludin (Ocln) Sigma-Aldrich SAB3500301 1:500 19 Mouse collagen, type IV Corning 354233 890 mg/ml 0.05 N HCl 100 μl -80 °C 890 mg/ml 50 μg/ml 56 μl collagen IV + 944 μl 0.05 N HCl 20 Dextran MW 60,000-90,000 Alfa Aesar J14495 1 kg RT 18% 5.4 g dextran in 30 ml DPBS 21 Penicillin/streptomycin (100x) Biochrom A2212 100 ml 1 ml -20 °C 100x 1x 100 μl pen/strep in 10 ml medium/dextran 22 L-glutamine Thermo Fisher Scientific GibcoTM 25030024 100 ml 1 ml -20 °C 200 mM (100x) 2 mM (1x) 100 μl L-glutamine in 10 ml medium 23 DMEM-F12 w/o glutamine Thermo Fisher Scientific GibcoTM 21331020 500 ml 4 °C 1x 1x undiluted 24 DMEM w/o glucose Thermo Fisher Scientific GibcoTM 11966025 500 ml 4 °C 1x 1x undiluted 25 Plasma-derived bovine serum (PDS) First Link 60-00-810 500 ml 10 ml -20 °C 100% 20% 10 ml PDS in 50 ml medium 26 Antibiotic/antimycotic (100x) Thermo Fisher Scientific GibcoTM 15240062 100 ml 1 ml -20 °C 100x 1x 100 μl AA in 10 ml medium/dextran 27 Heparin-sodium Ratiopharm PZN 003029843 1 ampule 4 °C 5,000 I.E./ml 750 I.E./50 ml 150 μl heparin in 50 ml medium 28 Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759 15 mg/5 ml DPBS 500 μl -20 °C 3 mg/ml 30 μg/ml 500 μl ECGS in 50 ml medium 29 18% dextran solution (see Recipes ) 30 Working medium (see Recipes ) 31 Digestion medium (see Recipes ) 32 Full medium (see Recipes ) Open in a separate window Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight.

Techniques: Concentration Assay, Sterility, Clinical Proteomics