mdck madin darby canine kidney ii cells (ATCC)
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mdck madin darby canine kidney ii cells
Mdck Madin Darby Canine Kidney Ii Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 7142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/cell+cloning-based+transcriptome+analysis/MDCK/pmc09768590-0-7-14
Average 99 stars, based on 7142 article reviews
Mdck Madin Darby Canine Kidney Ii Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 7142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/cell+cloning-based+transcriptome+analysis/MDCK/pmc09768590-0-7-14
Average 99 stars, based on 7142 article reviews
mdck madin darby canine kidney ii cells - by Bioz Stars,
2026-10
99/100 stars
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1) Product Images from "Sonic hedgehog is basolaterally sorted from the TGN and transcytosed to the apical domain involving Dispatched-1 at Rab11-ARE"
Article Title: Sonic hedgehog is basolaterally sorted from the TGN and transcytosed to the apical domain involving Dispatched-1 at Rab11-ARE
Journal: Frontiers in Cell and Developmental Biology
doi: 10.3389/fcell.2022.833175
Figure Legend Snippet: Shh is apically distributed and secreted in stable transfected MDCK cells. (A) Domain-selective biotinylation of stable transfected MDCK-Shh grown in Transwell filters. MDCK-Shh clone-1 shows Shh mainly at the apical cell surface, changing to non-polarized when cells are treated with 2 mM Na + -butyrate for 24 h to increase Shh expression. MDCK-Shh clone-2 expressing higher Shh levels than clone-1 displays Shh equally distributed among the apical and basolateral cell surface. E-cadherin and Na + /K + -ATPase basolateral distribution corroborates the polarized status of the cells. (B) Graphs show ∼80% apical cell surface distribution (n = 6) and more than 90% apical secretion of Shh in 6 h conditioned media in MDCK-Shh clone-1 (n = 3), (*** p < 0.001); (C) Polarized MDCK-Shh clone 1 cells immunostained for apical cell surface Shh in non-permeabilized cells grown in filters, as well as for total Shh (green) and E-cadherin (magenta) in cells grown in glass coverslips, as indicated. Images are confocal z-stacks maximum projections. Scale bar, 10 μm.
Techniques Used: Transfection, Expressing
Figure Legend Snippet: Distribution of Disp-1 in polarized MDCK cells. (A) Endogenous Disp-1 expression in MDCK cells. RT-PCR shows the expected band of 562 bp according to primers (see Materials and Methods). (B) MDCK cells grown in glass coverslips or Transwell filters for 3–4 days after reaching confluence were microinjected with expression plasmids for Shh and LDLR-GFP-Y18A, incubated for 4 h at 37°C and processed to detect each expressed protein. Indirect immunofluorescence of Shh (green) and direct imaging of LDLR-GFP-Y18A (magenta) in permeabilized cells show a similar apical and basolateral distribution, respectively, in glass coverslips and filters, validating the polarity conditions. (C) Rab11-ARE distribution in non-polarized and polarized MDCK cells. MDCK cells stably transfected with Rab11-CFP cDNA were grown on glass coverslips until confluence (left) or 3–4 days after confluence (right), then fixed, and imaged by confocal microscopy. The characteristic disperse perinuclear distribution is shown in non-polarized cells, whereas the punctate-like subapical localization is observed after 3–4 days of confluence in coverslips. (D) Distribution of Shh relative to Disp-1 expressed by plasmid microinjection. Polarized MDCK cells in glass coverslips were microinjected to coexpress Shh and Disp-1, fixed and immuno-stained by the apical side with anti-Shh (green) antibody, and then permeabilized and stained for Disp-1 with anti-HA (magenta) antibodies. Disp-1 mainly distributes underneath the apical cell surface stained by non-permeabilized anti-Shh antiody. (E) Polarized MDCK cells grown and treated as in (D) , were fixed, permeabilized, and stained for total anti-Shh (green) and anti-HA (magenta), showing similar Disp-1 and Shh distribution as in (D) . Scale bar, 10 μm.
Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Immunofluorescence, Imaging, Stable Transfection, Transfection, Confocal Microscopy, Plasmid Preparation, Microinjection, Staining
Figure Legend Snippet: Apical sorting of newly synthesized Shh is indirect through transcytosis. (A) Cell surface targeting assay of newly synthesized Shh and E-cadherin in polarized MDCK-Shh cells grown in Transwell filters. The cells were pulse-labeled with 35 S-methionine/cysteine and chased at 37°C for the indicated times by subsequent cell-surface-specific biotinylation at 4°C. S 35 -Shh is first detected at the basolateral cell surface and then gradually increased at the apical domain, while S 35 -E-cadherin remains basolateral. Graph shows the relative distribution of S 35 -Shh at different time points (30′, n = 5; 60′, n = 7; 120′, n = 4; and 180′, n = 2). (B) Cell surface targeting assay of newly synthesized GPI-anchored protein (GFP-NO-GPI) shows direct apical sorting. (C) Shh basolateral-to-apical transcytosis assay. Filter-grown MDCK-Shh cells were basolaterally labeled at 4°C with reducible biotin linkage (NHS-SS-Biotin), incubated at 37°C for 120 min and then subjected to MesNa reduction at apical or basolateral sides. Shh-biotin, but not E-cadherin–biotin, decreased by reduction at the apical side indicating Shh transcytosis. Graph shows percentage of Shh-biotin after reduction (n = 3; mean ± SEM; unpaired t -test; * p < 0.05). (D) Basolateral cell surface Shh becomes apically secreted. MDCK-Shh cells biotinylated from the basolateral side at 4°C and then incubated at 37°C for 2 h mainly release biotinylated Shh toward the apical media. Graph shows the percentage of total secreted biotinylated Shh precipitated with NeutrAvidin–Agarose beads followed by immunoblot (n = 4; mean ± SEM; unpaired t -test; *** p < 0.001).
Techniques Used: Synthesized, Labeling, Incubation, Western Blot
Figure Legend Snippet: Shh transcytosis. Polarized MDCK-Shh cells in Transwell filters were incubated with anti-Shh antibodies at 4°C from the basolateral (A – C) or apical (D , E) sides, shifted to 37°C for the indicated times, and detected by the secondary antibody. (A) Antibody-tagged basolateral Shh moved to the apical domain including a subapical punctate compartment, as shown by the secondary antibody (green) added after permeabilizing the cells at the indicated time points. (B) Shh (green) colocalization with endogenous Rab11 (red) after 30 and 60 min of trafficking. Graph shows the percentage of Rab11 colocalizing with Shh (mean ± SEM, n = 15 cells, unpaired t -test *** p < 0.001. (C) Apical cell surface detection of antibody-tagged basolateral Shh. Secondary antibody (red) was added to the apical side of non-permeabilized cells, which then were permeabilized and incubated with another secondary antibody (green). (D) Antibody-tagged apical Shh moved to the basolateral domain, as shown by total staining of permeabilized cells. (E) Basolateral cell surface detection of antibody-tagged apical Shh. Secondary antibody (red) was added to the basolateral side of non-permeabilized cells, which then were permeabilized and incubated with another secondary antibody (green). Scale bar, 10 μm.
Techniques Used: Incubation, Staining
Figure Legend Snippet: Sorting of Shh lipidation mutants. (A) Schematic Shh processing and lipidation. (B – F) Stable expression of MDCK cells indicated Shh lipidation mutants: lacking both lipids (ShhNC24S) (B) , only palmitoylated (ShhN) (C) , or only cholesteroylated (ShhNpC24S) (D) were analyzed by domain-specific cell surface biotinylation and secretion. Basolateral E-cadherin and Na + /K + ATPase transmembrane proteins and secreted fibronectin (FN) show cell polarity. (E) MDCK-ShhNpC24S cells were pulse-labeled with S 35 -methionine/cysteine and chased to detect protein arrival to each cell surface along with its polarized secretion. Graph shows the mean±SEM from two independent experiments. (F) Secretion of transcytosed ShhNpC24S. MDCK-ShhNpC24S cells biotinylated from the basolateral side at 4°C were then incubated at 37°C for 2 h, and immunoblot of secreted ShhNpC24S was performed on apical and basolateral conditioned media. Graphs of biotinylation and secretion assays (n = 3) represent mean±SEM. ** p < 0.05; *** p < 0.001, ns = no significance.
Techniques Used: Expressing, Labeling, Incubation, Western Blot
Figure Legend Snippet: Distribution of newly synthesized Shh when co-expressed with Disp-1 or Disp-1-CS. (A) Polarized MDCK cells in glass coverslips were microinjected to express Shh alone (upper panels), together with Disp-1 (middle panels), or Disp-1-CS (lower panels). Images of indirect immunofluorescence for Shh (green) and Disp-1 proteins (HA, magenta) are shown in maximal projections with zoomed images including basolateral (BL) views at the right. When coexpressed with Disp-1, Shh is not only distributed at the apical domain but also appears at basolateral borders of non-microinjected neighboring cells (arrow heads), reflecting its increased secretion. Meanwhile, when coexpressed with Disp-1-CS, Shh became enriched in a punctate subapical compartment (arrow heads) reminiscent of Rab11-ARE. (B) Colocalization of Rab11 with Shh and Disp-1. Polarized MDCK cells in glass coverslips were microinjected with Shh and Disp-1 plasmids, allowed for expression for 4 h at 37°C, fixed, permeabilized, and immunostained for Shh (green), Disp-1 or Disp-1-CS (magenta), and endogenous Rab11 (yellow). (C) Graphs quantified Rab11 colocalization with each protein in cells coexpressing either Disp-1 or Disp-1-CS, as indicated. Rab11 colocalized more with coexpressed Disp-1-CS than Disp-1 (left graph). Co-expressed Disp-1-CS increased Rab11 colocalization with Shh (right graph) (mean±SEM, n = 25 cells from at least three different experiments). *** p < 0.001. Scale bar, 10 μm.
Techniques Used: Synthesized, Immunofluorescence, Expressing
Figure Legend Snippet: High-resolution image of subapical endosomes containing Shh, Disp-1, and Rab11. Polarized MDCK cells in glass coverslips were microinjected to express Shh and Disp-1 for 4h, fixed, permeabilized, and processed for indirect immunofluorescence of Rab11 (yellow), Shh (green), and Disp-1 (magenta), and 12 confocal images of 100 nm thickness were acquired at the subapical region that include the Rab11 compartment. Deconvolved images are shown as maximum projections (upper panels) and as surface 3D-rendered reconstruction of apical planes (lower panels).
Techniques Used: Immunofluorescence
Figure Legend Snippet: Live-cell imaging of Shh-GFP in cells coexpressing Disp-1 or Disp-1-CS. Polarized MDCK cells in glass coverslips were microinjected to express Shh-GFP alone or together with Disp-1 or Disp-1-CS. (A) Shh-GFP shows apical distribution after 4 h of expression. (B) Polarized MDCK cells stably expressing Rab11-CFP were microinjected with Shh-GFP plasmid and after 45 min of expression at 37°C, the cells were incubated for 2 h at 20°C to arrest trafficking of protein at the TGN (time = 0) and then shifted to 37°C to reestablish exit from the TGN for 90 min. Arrow heads show basolaterally arrived Shh-GFP, while arrows point to Rab11-ARE showing colocalization with Shh-GFP very likely reflecting its transcytotic route to the apical domain. (C) Polarized MDCK cells microinjected with indicated plasmids and treated as in B to accumulate Shh-GFP at the TGN were analyzed by live-cell imaging for the indicated times. Shh-GFP detected in the border of neighboring non-expressing cells reflects its basolateral secretion and diffusion, which was enhanced by co-expressed Disp-1 but not Disp-1-CS, as more clearly seen in the overexposed 120 min image. (D) Quantification of Shh fluorescence relative intensity at the cell borders of microinjected cells and their neighbors. A line was drawn from the center of an injected cell toward the surrounding non-injected cells. Plot-profile of the line measuring the fluorescence intensity shows peaks revealing cell borders, which are depicted in the graph as intensities relative to injected cells. Scale bar, 10 μm.
Techniques Used: Live Cell Imaging, Expressing, Stable Transfection, Plasmid Preparation, Incubation, Diffusion-based Assay, Fluorescence, Injection
Figure Legend Snippet: Key resources table.
Techniques Used: Microinjection, Live Cell Imaging, Transfection, Construct, PCR Cloning, Stable Transfection, Expressing, Transduction, Software, Western Blot, Immunofluorescence
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Article Snippet: Em grade LFG distribution Cat# EM-15713 Crystal Violet Sigma Cat# C3886 Ethylenediaminetetraacetic acid (EDTA) Sigma Aldrich Cat# E9884 Methanol VWR Cat#20856.296 Ethanol absolute VWR Cat#20821.296P Trizol Thermo Fischer Scientific Cat#15596026 Tri Reagent Euromedex Cat#TR118-100 Thiazolyl Blue Tetrazolium Bromide (MTT) Sigma Aldrich Cat# M2128 Tris-HCl Sigma Aldrich Cat# T5941 Glycerol Sigma Aldrich Cat# G5516 Bovine serum albumin (BSA) Sigma Aldrich Cat# A8806 Triton X-100 Sigma Aldrich Cat# X100-100ML Liberase, Roche Cat#05401020001 Dispase II Roche Cat#4942078001 DNAse Roche Cat#11284932001 Baloxavir marboxil MedChemExpress Cat#HY-109025A TPCK-treated trypsin Sigma Aldrich Cat#T1426-MG Critical commercial assays Celsior solution Institut Georges Lopez Cat#533682 iScript cDNA Synthesis kit Bio-Rad Cat#1708891BUN High-Capacity RNA-to-cDNA kit Thermo Fisher Scientific Cat# 4368814 Platinum SYBR Green qPCR SuperMix-UDG Invitrogen Cat# 11733046 Direct-zol RNA Microprep Kits Zymo Cat# R2062 LDH-Glo Cytotoxicity assay Promega Cat# J2380 Cytometric Bead Array (CBA) Master Buffet Kit BD Biosciences Cat#558265 CBA Assay Diluent BD Biosciences Cat#560104 BD CBA Human IL-8 Flex Set BD Biosciences Cat#558277 BD CBA Human IP-10 Flex Set BD Biosciences Cat#558280 BD CBA Human IL-6 Flex Set BD Biosciences Cat#558276 BD CBA Human MCP-1 Flex Set BD Biosciences Cat#558287 Experimental models: Cell lines Crocin Bleaching Assay:Article Title: Agarose-free protocol for preparing human organotypic lung cultures to study respiratory virus infection and evaluate antivirals ex vivo. Article Snippet: Em grade LFG distribution Cat# EM-15713 Crystal Violet Sigma Cat# C3886 Ethylenediaminetetraacetic acid (EDTA) Sigma Aldrich Cat# E9884 Methanol VWR Cat#20856.296 Ethanol absolute VWR Cat#20821.296P Trizol Thermo Fischer Scientific Cat#15596026 Tri Reagent Euromedex Cat#TR118-100 Thiazolyl Blue Tetrazolium Bromide (MTT) Sigma Aldrich Cat# M2128 Tris-HCl Sigma Aldrich Cat# T5941 Glycerol Sigma Aldrich Cat# G5516 Bovine serum albumin (BSA) Sigma Aldrich Cat# A8806 Triton X-100 Sigma Aldrich Cat# X100-100ML Liberase, Roche Cat#05401020001 Dispase II Roche Cat#4942078001 DNAse Roche Cat#11284932001 Baloxavir marboxil MedChemExpress Cat#HY-109025A TPCK-treated trypsin Sigma Aldrich Cat#T1426-MG Critical commercial assays Celsior solution Institut Georges Lopez Cat#533682 iScript cDNA Synthesis kit Bio-Rad Cat#1708891BUN High-Capacity RNA-to-cDNA kit Thermo Fisher Scientific Cat# 4368814 Platinum SYBR Green qPCR SuperMix-UDG Invitrogen Cat# 11733046 Direct-zol RNA Microprep Kits Zymo Cat# R2062 LDH-Glo Cytotoxicity assay Promega Cat# J2380 Cytometric Bead Array (CBA) Master Buffet Kit BD Biosciences Cat#558265 CBA Assay Diluent BD Biosciences Cat#560104 BD CBA Human IL-8 Flex Set BD Biosciences Cat#558277 BD CBA Human IP-10 Flex Set BD Biosciences Cat#558280 BD CBA Human IL-6 Flex Set BD Biosciences Cat#558276 BD CBA Human MCP-1 Flex Set BD Biosciences Cat#558287 Experimental models: Cell lines Software:Article Title: Agarose-free protocol for preparing human organotypic lung cultures to study respiratory virus infection and evaluate antivirals ex vivo. Article Snippet: Em grade LFG distribution Cat# EM-15713 Crystal Violet Sigma Cat# C3886 Ethylenediaminetetraacetic acid (EDTA) Sigma Aldrich Cat# E9884 Methanol VWR Cat#20856.296 Ethanol absolute VWR Cat#20821.296P Trizol Thermo Fischer Scientific Cat#15596026 Tri Reagent Euromedex Cat#TR118-100 Thiazolyl Blue Tetrazolium Bromide (MTT) Sigma Aldrich Cat# M2128 Tris-HCl Sigma Aldrich Cat# T5941 Glycerol Sigma Aldrich Cat# G5516 Bovine serum albumin (BSA) Sigma Aldrich Cat# A8806 Triton X-100 Sigma Aldrich Cat# X100-100ML Liberase, Roche Cat#05401020001 Dispase II Roche Cat#4942078001 DNAse Roche Cat#11284932001 Baloxavir marboxil MedChemExpress Cat#HY-109025A TPCK-treated trypsin Sigma Aldrich Cat#T1426-MG Critical commercial assays Celsior solution Institut Georges Lopez Cat#533682 iScript cDNA Synthesis kit Bio-Rad Cat#1708891BUN High-Capacity RNA-to-cDNA kit Thermo Fisher Scientific Cat# 4368814 Platinum SYBR Green qPCR SuperMix-UDG Invitrogen Cat# 11733046 Direct-zol RNA Microprep Kits Zymo Cat# R2062 LDH-Glo Cytotoxicity assay Promega Cat# J2380 Cytometric Bead Array (CBA) Master Buffet Kit BD Biosciences Cat#558265 CBA Assay Diluent BD Biosciences Cat#560104 BD CBA Human IL-8 Flex Set BD Biosciences Cat#558277 BD CBA Human IP-10 Flex Set BD Biosciences Cat#558280 BD CBA Human IL-6 Flex Set BD Biosciences Cat#558276 BD CBA Human MCP-1 Flex Set BD Biosciences Cat#558287 Experimental models: Cell lines |