Virus:Article Title: Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.
Article Snippet: AbCam Cat# ab30522; RRID: AB_732219 Rat Anti-Mouse CD45 (clone 30-F11), BUV 395 BD Biosciences Cat# 564279; RRID: AB_2651134 beta Actin loading control Monoclonal Antibody (clone BA3R) Thermo Fisher Scientific Cat# MA5-15739; RRID: AB_10979409 (Continued on next page) Cell 170, 1–15.e1–e8, August 24, 2017 e2 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022 .. REAGENT or RESOURCE SOURCE IDENTIFIER Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# R37118; RRID: AB_2556546 AF568-conjugated phalloidin Thermo Fisher Scientific Cat# A12380 AF488 conjugated polyclonal rabbit anti-GFP Thermo Fisher Scientific Cat# A21311 Goat anti-Rabbit IgG (H+L) Secondary Antibody, Pacific Blue Invitrogen Cat# P-10994; RRID: AB_2539814 Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) Cell Signaling Technology Cat# 5366 Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) Cell Signaling Technology Cat# 5151 Bacterial and Virus Strains Uropathogenic E. coli (UPEC, UT189) Hung et al., 2009 (a gift from S. Hultgren) N/A Biological Samples Human kidney samples (ethical approval REC12/EE/0446) NHSBT N/A Human whole blood samples (ethical approval REC08/H0308/176) National Blood Service Cat# Leukocyte cone NC24 Chemicals, Peptides, and Recombinant Proteins Recombinant human CX3CL1 Life Technologies Cat# 10636H08H50 Recombinant human CCL2 R & D Systems Cat# 279-MC-050 0-120 mmol/L sodium chloride South Devon Healthcare Cat# Sodium Chloride 30% w/v Concentrate 0-200 mOsmol/kg 20% mannitol Baxter Cat# mannitol 100 ng/ml E. coli lipopolysaccharide Sigma-Aldrich Cat# L2630 p38 inhibitor SB203580 Cell signaling technology Cat# SB203580 5633 Bortezomib (PS-341) proteasome inhibitor SelleckChem Cat# S1013 Critical Commercial Assays FoxP3 intracellular staining kit eBioscience Cat# 00-5521-00 CD11c bead separation BD Biosciences Cat# 130-097-059 MACS separation using LS columns Miltenyi Botech Cat# 130-042-401 Complementary DNA synthesis; High capacity RNA to cDNA Life Technologies Cat# 4387406 NFAT5 siRNA knockdown (using Lipofectamine and silencer select siRNA) Thermo Fisher Scientific Lipofectamine Cat# 18324012; siRNA Cat# AM16708 Deposited Data Paired human cortex and medulla kidney samples Higgins et al., 2004 Gene Expression Omnibus (GSE3931) Experimental Models: Cell Lines Renal tubular epithelial cells (HK2 cells) ATCC Cat# CRL-2190; RRID:CVCL_0302 Experimental Models: Organisms/Strains Mouse: Wild-type C57BL/6 Jackson Laboratories Stock No: 000664 Mouse: UBI-GFP-BL/6 mice. .. Strain ID: C57BL/6-Tg(UBC-GFP)30Scha/J Jackson Laboratories Stock No: 004353 Mouse: CD11c EYFP Strain ID: C.Cg-Tg(Itgax-Venus)1Mnz/J Gift from M. Nussenzweig Stock No: 008830 (Jackson Laboratories) Mouse: Ccr2 / Strain ID: B6.129S4Ccr2tm1Ifc/J Boring et al., 1997 (Gift from Callum Bain and Simon Jenkins) Stock No: 004999 (Jackson Laboratories) (Continued on next page) e3 Cell 170, 1–15.e1–e8, August 24, 2017 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022
Recombinant:Article Title: Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.
Article Snippet: AbCam Cat# ab30522; RRID: AB_732219 Rat Anti-Mouse CD45 (clone 30-F11), BUV 395 BD Biosciences Cat# 564279; RRID: AB_2651134 beta Actin loading control Monoclonal Antibody (clone BA3R) Thermo Fisher Scientific Cat# MA5-15739; RRID: AB_10979409 (Continued on next page) Cell 170, 1–15.e1–e8, August 24, 2017 e2 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022 .. REAGENT or RESOURCE SOURCE IDENTIFIER Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# R37118; RRID: AB_2556546 AF568-conjugated phalloidin Thermo Fisher Scientific Cat# A12380 AF488 conjugated polyclonal rabbit anti-GFP Thermo Fisher Scientific Cat# A21311 Goat anti-Rabbit IgG (H+L) Secondary Antibody, Pacific Blue Invitrogen Cat# P-10994; RRID: AB_2539814 Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) Cell Signaling Technology Cat# 5366 Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) Cell Signaling Technology Cat# 5151 Bacterial and Virus Strains Uropathogenic E. coli (UPEC, UT189) Hung et al., 2009 (a gift from S. Hultgren) N/A Biological Samples Human kidney samples (ethical approval REC12/EE/0446) NHSBT N/A Human whole blood samples (ethical approval REC08/H0308/176) National Blood Service Cat# Leukocyte cone NC24 Chemicals, Peptides, and Recombinant Proteins Recombinant human CX3CL1 Life Technologies Cat# 10636H08H50 Recombinant human CCL2 R & D Systems Cat# 279-MC-050 0-120 mmol/L sodium chloride South Devon Healthcare Cat# Sodium Chloride 30% w/v Concentrate 0-200 mOsmol/kg 20% mannitol Baxter Cat# mannitol 100 ng/ml E. coli lipopolysaccharide Sigma-Aldrich Cat# L2630 p38 inhibitor SB203580 Cell signaling technology Cat# SB203580 5633 Bortezomib (PS-341) proteasome inhibitor SelleckChem Cat# S1013 Critical Commercial Assays FoxP3 intracellular staining kit eBioscience Cat# 00-5521-00 CD11c bead separation BD Biosciences Cat# 130-097-059 MACS separation using LS columns Miltenyi Botech Cat# 130-042-401 Complementary DNA synthesis; High capacity RNA to cDNA Life Technologies Cat# 4387406 NFAT5 siRNA knockdown (using Lipofectamine and silencer select siRNA) Thermo Fisher Scientific Lipofectamine Cat# 18324012; siRNA Cat# AM16708 Deposited Data Paired human cortex and medulla kidney samples Higgins et al., 2004 Gene Expression Omnibus (GSE3931) Experimental Models: Cell Lines Renal tubular epithelial cells (HK2 cells) ATCC Cat# CRL-2190; RRID:CVCL_0302 Experimental Models: Organisms/Strains Mouse: Wild-type C57BL/6 Jackson Laboratories Stock No: 000664 Mouse: UBI-GFP-BL/6 mice. .. Strain ID: C57BL/6-Tg(UBC-GFP)30Scha/J Jackson Laboratories Stock No: 004353 Mouse: CD11c EYFP Strain ID: C.Cg-Tg(Itgax-Venus)1Mnz/J Gift from M. Nussenzweig Stock No: 008830 (Jackson Laboratories) Mouse: Ccr2 / Strain ID: B6.129S4Ccr2tm1Ifc/J Boring et al., 1997 (Gift from Callum Bain and Simon Jenkins) Stock No: 004999 (Jackson Laboratories) (Continued on next page) e3 Cell 170, 1–15.e1–e8, August 24, 2017 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022
Staining:Article Title: Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.
Article Snippet: AbCam Cat# ab30522; RRID: AB_732219 Rat Anti-Mouse CD45 (clone 30-F11), BUV 395 BD Biosciences Cat# 564279; RRID: AB_2651134 beta Actin loading control Monoclonal Antibody (clone BA3R) Thermo Fisher Scientific Cat# MA5-15739; RRID: AB_10979409 (Continued on next page) Cell 170, 1–15.e1–e8, August 24, 2017 e2 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022 .. REAGENT or RESOURCE SOURCE IDENTIFIER Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# R37118; RRID: AB_2556546 AF568-conjugated phalloidin Thermo Fisher Scientific Cat# A12380 AF488 conjugated polyclonal rabbit anti-GFP Thermo Fisher Scientific Cat# A21311 Goat anti-Rabbit IgG (H+L) Secondary Antibody, Pacific Blue Invitrogen Cat# P-10994; RRID: AB_2539814 Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) Cell Signaling Technology Cat# 5366 Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) Cell Signaling Technology Cat# 5151 Bacterial and Virus Strains Uropathogenic E. coli (UPEC, UT189) Hung et al., 2009 (a gift from S. Hultgren) N/A Biological Samples Human kidney samples (ethical approval REC12/EE/0446) NHSBT N/A Human whole blood samples (ethical approval REC08/H0308/176) National Blood Service Cat# Leukocyte cone NC24 Chemicals, Peptides, and Recombinant Proteins Recombinant human CX3CL1 Life Technologies Cat# 10636H08H50 Recombinant human CCL2 R & D Systems Cat# 279-MC-050 0-120 mmol/L sodium chloride South Devon Healthcare Cat# Sodium Chloride 30% w/v Concentrate 0-200 mOsmol/kg 20% mannitol Baxter Cat# mannitol 100 ng/ml E. coli lipopolysaccharide Sigma-Aldrich Cat# L2630 p38 inhibitor SB203580 Cell signaling technology Cat# SB203580 5633 Bortezomib (PS-341) proteasome inhibitor SelleckChem Cat# S1013 Critical Commercial Assays FoxP3 intracellular staining kit eBioscience Cat# 00-5521-00 CD11c bead separation BD Biosciences Cat# 130-097-059 MACS separation using LS columns Miltenyi Botech Cat# 130-042-401 Complementary DNA synthesis; High capacity RNA to cDNA Life Technologies Cat# 4387406 NFAT5 siRNA knockdown (using Lipofectamine and silencer select siRNA) Thermo Fisher Scientific Lipofectamine Cat# 18324012; siRNA Cat# AM16708 Deposited Data Paired human cortex and medulla kidney samples Higgins et al., 2004 Gene Expression Omnibus (GSE3931) Experimental Models: Cell Lines Renal tubular epithelial cells (HK2 cells) ATCC Cat# CRL-2190; RRID:CVCL_0302 Experimental Models: Organisms/Strains Mouse: Wild-type C57BL/6 Jackson Laboratories Stock No: 000664 Mouse: UBI-GFP-BL/6 mice. .. Strain ID: C57BL/6-Tg(UBC-GFP)30Scha/J Jackson Laboratories Stock No: 004353 Mouse: CD11c EYFP Strain ID: C.Cg-Tg(Itgax-Venus)1Mnz/J Gift from M. Nussenzweig Stock No: 008830 (Jackson Laboratories) Mouse: Ccr2 / Strain ID: B6.129S4Ccr2tm1Ifc/J Boring et al., 1997 (Gift from Callum Bain and Simon Jenkins) Stock No: 004999 (Jackson Laboratories) (Continued on next page) e3 Cell 170, 1–15.e1–e8, August 24, 2017 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022
Magnetic Cell Separation:Article Title: Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.
Article Snippet: AbCam Cat# ab30522; RRID: AB_732219 Rat Anti-Mouse CD45 (clone 30-F11), BUV 395 BD Biosciences Cat# 564279; RRID: AB_2651134 beta Actin loading control Monoclonal Antibody (clone BA3R) Thermo Fisher Scientific Cat# MA5-15739; RRID: AB_10979409 (Continued on next page) Cell 170, 1–15.e1–e8, August 24, 2017 e2 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022 .. REAGENT or RESOURCE SOURCE IDENTIFIER Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# R37118; RRID: AB_2556546 AF568-conjugated phalloidin Thermo Fisher Scientific Cat# A12380 AF488 conjugated polyclonal rabbit anti-GFP Thermo Fisher Scientific Cat# A21311 Goat anti-Rabbit IgG (H+L) Secondary Antibody, Pacific Blue Invitrogen Cat# P-10994; RRID: AB_2539814 Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) Cell Signaling Technology Cat# 5366 Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) Cell Signaling Technology Cat# 5151 Bacterial and Virus Strains Uropathogenic E. coli (UPEC, UT189) Hung et al., 2009 (a gift from S. Hultgren) N/A Biological Samples Human kidney samples (ethical approval REC12/EE/0446) NHSBT N/A Human whole blood samples (ethical approval REC08/H0308/176) National Blood Service Cat# Leukocyte cone NC24 Chemicals, Peptides, and Recombinant Proteins Recombinant human CX3CL1 Life Technologies Cat# 10636H08H50 Recombinant human CCL2 R & D Systems Cat# 279-MC-050 0-120 mmol/L sodium chloride South Devon Healthcare Cat# Sodium Chloride 30% w/v Concentrate 0-200 mOsmol/kg 20% mannitol Baxter Cat# mannitol 100 ng/ml E. coli lipopolysaccharide Sigma-Aldrich Cat# L2630 p38 inhibitor SB203580 Cell signaling technology Cat# SB203580 5633 Bortezomib (PS-341) proteasome inhibitor SelleckChem Cat# S1013 Critical Commercial Assays FoxP3 intracellular staining kit eBioscience Cat# 00-5521-00 CD11c bead separation BD Biosciences Cat# 130-097-059 MACS separation using LS columns Miltenyi Botech Cat# 130-042-401 Complementary DNA synthesis; High capacity RNA to cDNA Life Technologies Cat# 4387406 NFAT5 siRNA knockdown (using Lipofectamine and silencer select siRNA) Thermo Fisher Scientific Lipofectamine Cat# 18324012; siRNA Cat# AM16708 Deposited Data Paired human cortex and medulla kidney samples Higgins et al., 2004 Gene Expression Omnibus (GSE3931) Experimental Models: Cell Lines Renal tubular epithelial cells (HK2 cells) ATCC Cat# CRL-2190; RRID:CVCL_0302 Experimental Models: Organisms/Strains Mouse: Wild-type C57BL/6 Jackson Laboratories Stock No: 000664 Mouse: UBI-GFP-BL/6 mice. .. Strain ID: C57BL/6-Tg(UBC-GFP)30Scha/J Jackson Laboratories Stock No: 004353 Mouse: CD11c EYFP Strain ID: C.Cg-Tg(Itgax-Venus)1Mnz/J Gift from M. Nussenzweig Stock No: 008830 (Jackson Laboratories) Mouse: Ccr2 / Strain ID: B6.129S4Ccr2tm1Ifc/J Boring et al., 1997 (Gift from Callum Bain and Simon Jenkins) Stock No: 004999 (Jackson Laboratories) (Continued on next page) e3 Cell 170, 1–15.e1–e8, August 24, 2017 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022
DNA Synthesis:Article Title: Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.
Article Snippet: AbCam Cat# ab30522; RRID: AB_732219 Rat Anti-Mouse CD45 (clone 30-F11), BUV 395 BD Biosciences Cat# 564279; RRID: AB_2651134 beta Actin loading control Monoclonal Antibody (clone BA3R) Thermo Fisher Scientific Cat# MA5-15739; RRID: AB_10979409 (Continued on next page) Cell 170, 1–15.e1–e8, August 24, 2017 e2 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022 .. REAGENT or RESOURCE SOURCE IDENTIFIER Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# R37118; RRID: AB_2556546 AF568-conjugated phalloidin Thermo Fisher Scientific Cat# A12380 AF488 conjugated polyclonal rabbit anti-GFP Thermo Fisher Scientific Cat# A21311 Goat anti-Rabbit IgG (H+L) Secondary Antibody, Pacific Blue Invitrogen Cat# P-10994; RRID: AB_2539814 Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) Cell Signaling Technology Cat# 5366 Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) Cell Signaling Technology Cat# 5151 Bacterial and Virus Strains Uropathogenic E. coli (UPEC, UT189) Hung et al., 2009 (a gift from S. Hultgren) N/A Biological Samples Human kidney samples (ethical approval REC12/EE/0446) NHSBT N/A Human whole blood samples (ethical approval REC08/H0308/176) National Blood Service Cat# Leukocyte cone NC24 Chemicals, Peptides, and Recombinant Proteins Recombinant human CX3CL1 Life Technologies Cat# 10636H08H50 Recombinant human CCL2 R & D Systems Cat# 279-MC-050 0-120 mmol/L sodium chloride South Devon Healthcare Cat# Sodium Chloride 30% w/v Concentrate 0-200 mOsmol/kg 20% mannitol Baxter Cat# mannitol 100 ng/ml E. coli lipopolysaccharide Sigma-Aldrich Cat# L2630 p38 inhibitor SB203580 Cell signaling technology Cat# SB203580 5633 Bortezomib (PS-341) proteasome inhibitor SelleckChem Cat# S1013 Critical Commercial Assays FoxP3 intracellular staining kit eBioscience Cat# 00-5521-00 CD11c bead separation BD Biosciences Cat# 130-097-059 MACS separation using LS columns Miltenyi Botech Cat# 130-042-401 Complementary DNA synthesis; High capacity RNA to cDNA Life Technologies Cat# 4387406 NFAT5 siRNA knockdown (using Lipofectamine and silencer select siRNA) Thermo Fisher Scientific Lipofectamine Cat# 18324012; siRNA Cat# AM16708 Deposited Data Paired human cortex and medulla kidney samples Higgins et al., 2004 Gene Expression Omnibus (GSE3931) Experimental Models: Cell Lines Renal tubular epithelial cells (HK2 cells) ATCC Cat# CRL-2190; RRID:CVCL_0302 Experimental Models: Organisms/Strains Mouse: Wild-type C57BL/6 Jackson Laboratories Stock No: 000664 Mouse: UBI-GFP-BL/6 mice. .. Strain ID: C57BL/6-Tg(UBC-GFP)30Scha/J Jackson Laboratories Stock No: 004353 Mouse: CD11c EYFP Strain ID: C.Cg-Tg(Itgax-Venus)1Mnz/J Gift from M. Nussenzweig Stock No: 008830 (Jackson Laboratories) Mouse: Ccr2 / Strain ID: B6.129S4Ccr2tm1Ifc/J Boring et al., 1997 (Gift from Callum Bain and Simon Jenkins) Stock No: 004999 (Jackson Laboratories) (Continued on next page) e3 Cell 170, 1–15.e1–e8, August 24, 2017 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022
Knockdown:Article Title: Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.
Article Snippet: AbCam Cat# ab30522; RRID: AB_732219 Rat Anti-Mouse CD45 (clone 30-F11), BUV 395 BD Biosciences Cat# 564279; RRID: AB_2651134 beta Actin loading control Monoclonal Antibody (clone BA3R) Thermo Fisher Scientific Cat# MA5-15739; RRID: AB_10979409 (Continued on next page) Cell 170, 1–15.e1–e8, August 24, 2017 e2 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022 .. REAGENT or RESOURCE SOURCE IDENTIFIER Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# R37118; RRID: AB_2556546 AF568-conjugated phalloidin Thermo Fisher Scientific Cat# A12380 AF488 conjugated polyclonal rabbit anti-GFP Thermo Fisher Scientific Cat# A21311 Goat anti-Rabbit IgG (H+L) Secondary Antibody, Pacific Blue Invitrogen Cat# P-10994; RRID: AB_2539814 Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) Cell Signaling Technology Cat# 5366 Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) Cell Signaling Technology Cat# 5151 Bacterial and Virus Strains Uropathogenic E. coli (UPEC, UT189) Hung et al., 2009 (a gift from S. Hultgren) N/A Biological Samples Human kidney samples (ethical approval REC12/EE/0446) NHSBT N/A Human whole blood samples (ethical approval REC08/H0308/176) National Blood Service Cat# Leukocyte cone NC24 Chemicals, Peptides, and Recombinant Proteins Recombinant human CX3CL1 Life Technologies Cat# 10636H08H50 Recombinant human CCL2 R & D Systems Cat# 279-MC-050 0-120 mmol/L sodium chloride South Devon Healthcare Cat# Sodium Chloride 30% w/v Concentrate 0-200 mOsmol/kg 20% mannitol Baxter Cat# mannitol 100 ng/ml E. coli lipopolysaccharide Sigma-Aldrich Cat# L2630 p38 inhibitor SB203580 Cell signaling technology Cat# SB203580 5633 Bortezomib (PS-341) proteasome inhibitor SelleckChem Cat# S1013 Critical Commercial Assays FoxP3 intracellular staining kit eBioscience Cat# 00-5521-00 CD11c bead separation BD Biosciences Cat# 130-097-059 MACS separation using LS columns Miltenyi Botech Cat# 130-042-401 Complementary DNA synthesis; High capacity RNA to cDNA Life Technologies Cat# 4387406 NFAT5 siRNA knockdown (using Lipofectamine and silencer select siRNA) Thermo Fisher Scientific Lipofectamine Cat# 18324012; siRNA Cat# AM16708 Deposited Data Paired human cortex and medulla kidney samples Higgins et al., 2004 Gene Expression Omnibus (GSE3931) Experimental Models: Cell Lines Renal tubular epithelial cells (HK2 cells) ATCC Cat# CRL-2190; RRID:CVCL_0302 Experimental Models: Organisms/Strains Mouse: Wild-type C57BL/6 Jackson Laboratories Stock No: 000664 Mouse: UBI-GFP-BL/6 mice. .. Strain ID: C57BL/6-Tg(UBC-GFP)30Scha/J Jackson Laboratories Stock No: 004353 Mouse: CD11c EYFP Strain ID: C.Cg-Tg(Itgax-Venus)1Mnz/J Gift from M. Nussenzweig Stock No: 008830 (Jackson Laboratories) Mouse: Ccr2 / Strain ID: B6.129S4Ccr2tm1Ifc/J Boring et al., 1997 (Gift from Callum Bain and Simon Jenkins) Stock No: 004999 (Jackson Laboratories) (Continued on next page) e3 Cell 170, 1–15.e1–e8, August 24, 2017 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022
Gene Expression:Article Title: Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.
Article Snippet: AbCam Cat# ab30522; RRID: AB_732219 Rat Anti-Mouse CD45 (clone 30-F11), BUV 395 BD Biosciences Cat# 564279; RRID: AB_2651134 beta Actin loading control Monoclonal Antibody (clone BA3R) Thermo Fisher Scientific Cat# MA5-15739; RRID: AB_10979409 (Continued on next page) Cell 170, 1–15.e1–e8, August 24, 2017 e2 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022 .. REAGENT or RESOURCE SOURCE IDENTIFIER Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# R37118; RRID: AB_2556546 AF568-conjugated phalloidin Thermo Fisher Scientific Cat# A12380 AF488 conjugated polyclonal rabbit anti-GFP Thermo Fisher Scientific Cat# A21311 Goat anti-Rabbit IgG (H+L) Secondary Antibody, Pacific Blue Invitrogen Cat# P-10994; RRID: AB_2539814 Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) Cell Signaling Technology Cat# 5366 Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) Cell Signaling Technology Cat# 5151 Bacterial and Virus Strains Uropathogenic E. coli (UPEC, UT189) Hung et al., 2009 (a gift from S. Hultgren) N/A Biological Samples Human kidney samples (ethical approval REC12/EE/0446) NHSBT N/A Human whole blood samples (ethical approval REC08/H0308/176) National Blood Service Cat# Leukocyte cone NC24 Chemicals, Peptides, and Recombinant Proteins Recombinant human CX3CL1 Life Technologies Cat# 10636H08H50 Recombinant human CCL2 R & D Systems Cat# 279-MC-050 0-120 mmol/L sodium chloride South Devon Healthcare Cat# Sodium Chloride 30% w/v Concentrate 0-200 mOsmol/kg 20% mannitol Baxter Cat# mannitol 100 ng/ml E. coli lipopolysaccharide Sigma-Aldrich Cat# L2630 p38 inhibitor SB203580 Cell signaling technology Cat# SB203580 5633 Bortezomib (PS-341) proteasome inhibitor SelleckChem Cat# S1013 Critical Commercial Assays FoxP3 intracellular staining kit eBioscience Cat# 00-5521-00 CD11c bead separation BD Biosciences Cat# 130-097-059 MACS separation using LS columns Miltenyi Botech Cat# 130-042-401 Complementary DNA synthesis; High capacity RNA to cDNA Life Technologies Cat# 4387406 NFAT5 siRNA knockdown (using Lipofectamine and silencer select siRNA) Thermo Fisher Scientific Lipofectamine Cat# 18324012; siRNA Cat# AM16708 Deposited Data Paired human cortex and medulla kidney samples Higgins et al., 2004 Gene Expression Omnibus (GSE3931) Experimental Models: Cell Lines Renal tubular epithelial cells (HK2 cells) ATCC Cat# CRL-2190; RRID:CVCL_0302 Experimental Models: Organisms/Strains Mouse: Wild-type C57BL/6 Jackson Laboratories Stock No: 000664 Mouse: UBI-GFP-BL/6 mice. .. Strain ID: C57BL/6-Tg(UBC-GFP)30Scha/J Jackson Laboratories Stock No: 004353 Mouse: CD11c EYFP Strain ID: C.Cg-Tg(Itgax-Venus)1Mnz/J Gift from M. Nussenzweig Stock No: 008830 (Jackson Laboratories) Mouse: Ccr2 / Strain ID: B6.129S4Ccr2tm1Ifc/J Boring et al., 1997 (Gift from Callum Bain and Simon Jenkins) Stock No: 004999 (Jackson Laboratories) (Continued on next page) e3 Cell 170, 1–15.e1–e8, August 24, 2017 Please cite this article in press as: Berry et al., Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone, Cell (2017), http:// dx.doi.org/10.1016/j.cell.2017.07.022
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