Transcriptomes of Major Proximal Tubule Cell Culture Models

November 12, 2020
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eaaci2015

Transcriptomes of Major Proximal Tubule Cell Culture Models

Background: Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology, and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend upon the genes expressed. New tools for transcriptomic profiling using RNA sequencing (RNA-Seq) make it possible to catalog expressed genes in each cell line.
Methods: Fourteen different proximal tubule cell lines, representing six species, were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures.
Results: Transcripts expressed in cell lines variably matched transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45% of proximal marker genes [TPM threshold =15]), with pig kidney cells (LLC-PK1) close behind (39%). Lower-percentage matches were seen for various human lines, including HK-2 (26%), and lines from rodent kidneys, such as NRK-52E (23%). Nominally, identical OK cells from different sources differed substantially in expression of proximal tubule markers. Mapping cell line transcriptomes to gene sets for various proximal tubule functions (sodium and water transport, protein transport, metabolic functions, endocrine functions) showed that different lines may be optimal for experimentally modeling each function.
An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created to interrogate cell line transcriptome data. Proteomic analysis of NRK-52E cells confirmed low expression of many proximal tubule marker proteins.
Conclusions: No cell line fully matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.

Fibroblast-like cells change gene expression of bone remodelling markers in transwell cultures

Introduction: Periprosthetic fibroblast-like cells (PPFs) play an important role in aseptic loosening of arthroplasties. Various studies have examined PPF behavior in monolayer culture systems. However, the periprosthetic tissue is a three-dimensional (3D) mesh, which allows the cells to interact in a multidirectional way. The expression of bone remodeling markers of fibroblast-like cells in a multilayer environment changes significantly versus monolayer cultures without the addition of particles or cytokine stimulation. Gene expression of bone remodeling markers was therefore compared in fibroblast-like cells from different origins and dermal fibroblasts under transwell culture conditions versus monolayer cultures.
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Methods: PPFs from periprosthetic tissues (n = 12), osteoarthritic (OA) synovial fibroblast-like cells (SFs) (n = 6), and dermal fibroblasts (DFs) were cultured in monolayer (density 5.5 × 103/cm2) or multilayer cultures (density 8.5 × 105/cm2) for 10 or 21 days. Cultures were examined via histology, TRAP staining, immunohistochemistry (anti-S100a4), and quantitative real-time PCR.
Results: Fibroblast-like cells (PPFs/SFs) and dermal fibroblasts significantly increased the expression of RANKL and significantly decreased the expression of ALP, COL1A1, and OPG in multilayer cultures. PPFs and SFs in multilayer cultures further showed a higher expression of cathepsin K, MMP-13, and TNF-α. In multilayer PPF cultures, the mRNA level of TRAP was also found to be significantly increased.
Conclusion: The multilayer cultures are able to induce significant expression changes in fibroblast-like cells depending on the nature of cellular origin without the addition of any further stimulus. This system might be a useful tool to get more in vivo like results regarding fibroblast-like cell cultures.

A novel method of cell culture based on the microfluidic chip for regulation of cell density

 

The regulation of cell density is an important segment in microfluidic cell culture, particularly in the repeated assays. Traditionally, the consistent cell density is difficult to achieve owing to the inaccurate regulation of cell density with manual feedback. A novel cell culture method with automatic feedback is proposed for real-time regulation of cell density based on microfluidic chip in this paper. Here, an integrated microfluidic system combining cell culture, density detection and control of proliferation rate was developed.

 

Interdigital electrode structures (IDES) were sputtered on the microchannel for automatically to provide the real-time feedback information of impedance. The most sensitive frequency was studied to improve the detection resolution of the sensing chip. Cells were cultured on the chip surface and cell density was detected by monitoring the alternation of the impedance. The feedback controller was established by least squares support vector machines (LS-SVM). Then, the cell proliferation rate was automatically controlled using the feedback controller to achieve the desired cell density in the repeated assays.

 

The results show that the standard error of this method is 2.8% indicating that the method can keep consistency of cell density in the repeated assays. This study provides a basis for improving the accuracy and repeatability in the further assays of finding the optimal drug concentration. This article is protected by copyright. All rights reserved.

 

Effects of Oxygen and Glucose on Bone Marrow Mesenchymal Stem Cell Culture

This study determines whether the viability of mesenchymal stem cell (MSC) in vitro is most sensitive to oxygen supply, energetic substrate supply, or accumulation of lactate. Mouse unmodified (wild type (WT)) and erythropoietin (EPO) gene-modified MSC is cultured for 7 days in normoxic (21%) and anoxic conditions. WT-MSC is cultured in anoxia for 45 days in high and regular glucose media and both have similar viability when compared to their normoxic controls at 7 days. Protein production of EPO-MSC is unaffected by the absence of oxygen.
MSC doubling time and post-anoxic exposure is increased (WT: 32.3-73.3 h; EPO: 27.2-115 h). High glucose leads to a 37% increase in cell viability at 13 days and 17% at 30 days, indicating that MSC anoxic survival is affected by supply of metabolic substrate. However, after 30 days, little difference in viability is found, and at 45 days, complete cell death occurs in both the conditions. This death cannot be attributed to lack of glucose or lactate levels. MSC stemness is retained for both osteogenic and adipogenic differentiations. The absence of oxygen increases the doubling time of MSC but does not affect their viability, protein production, or differentiation capacity.

Monolayer/spheroid co-culture of cells on a PDMS well plate mediated by selective polydopamine coating

Here, we have selectively coated polydopamine (PDA) onto a polydimethylsiloxane (PDMS) well plate to enable the cell co-culture of a monolayer and spheroids in a semi-segregated manner. During the coating process, the contact between the PDA solution and PDMS well plate was limited to the outer flat surface because the strong hydrophobicity of PDMS prevented the access of the PDA solution into the concave structures. This resulted in a spatially-defined coating of PDA. The success of PDA coating was evidenced by measuring the water contact angle, observing the liquid-air interface, and via PDA-specific metallization.
This platform provides a simultaneous cell culture in both a monolayer and spheroids employing either monotypic or heterotypic cells. For the monotypic culture, mesenchymal stem cells (MSCs) were seeded over the well plate to concurrently generate the monolayer and spheroids. In the heterotypic culture, MSCs were first seeded into the wells to form spheroids. Then, human umbilical vein endothelial cells (HUVECs) were added over the flat surface of the well plate and allowed to form a monolayer. The microscopic observation and fluorescence-based cell staining confirmed the clear segregation between the monolayer and spheroids in both monotypic and heterotypic cultures. This new model could pave the way for the construction of a platform closely mimicking the physiological environment used to investigate cell-cell interactions and communications applicable for drug screening.
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Description: The proteasome is a multicatalytic proteinase complex with a highly ordered ring-shaped 20S core structure. The core structure is composed of 4 rings of 28 non-identical subunits; 2 rings are composed of 7 alpha subunits and 2 rings are composed of 7 beta subunits. Proteasomes are distributed throughout eukaryotic cells at a high concentration and cleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway. An essential function of a modified proteasome, the immunoproteasome, is the processing of class I MHC peptides. This gene encodes a member of the proteasome B-type family, also known as the T1B family, that is a 20S core beta subunit. This gene is located in the class II region of the MHC (major histocompatibility complex). Expression of this gene is induced by gamma interferon and this gene product replaces catalytic subunit 3 (proteasome beta 5 subunit) in the immunoproteasome. Proteolytic processing is required to generate a mature subunit. Two alternative transcripts encoding two isoforms have been identified; both isoforms are processed to yield the same mature subunit.
Anti-PSMB8 antibody
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Description: The proteasome is a multicatalytic proteinase complex with a highly ordered ring-shaped 20S core structure. The core structure is composed of 4 rings of 28 non-identical subunits; 2 rings are composed of 7 alpha subunits and 2 rings are composed of 7 beta subunits. Proteasomes are distributed throughout eukaryotic cells at a high concentration and cleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway. An essential function of a modified proteasome, the immunoproteasome, is the processing of class I MHC peptides. This gene encodes a member of the proteasome B-type family, also known as the T1B family, that is a 20S core beta subunit. This gene is located in the class II region of the MHC (major histocompatibility complex). Expression of this gene is induced by gamma interferon and this gene product replaces catalytic subunit 3 (proteasome beta 5 subunit) in the immunoproteasome. Proteolytic processing is required to generate a mature subunit. Two alternative transcripts encoding two isoforms have been identified; both isoforms are processed to yield the same mature subunit.
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Description: A polyclonal antibody against PSMB8. Recognizes PSMB8 from Human. This antibody is HRP conjugated. Tested in the following application: ELISA
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Description: A polyclonal antibody against PSMB8. Recognizes PSMB8 from Human. This antibody is FITC conjugated. Tested in the following application: ELISA
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Description: A polyclonal antibody against PSMB8. Recognizes PSMB8 from Human. This antibody is Biotin conjugated. Tested in the following application: ELISA
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  • Shipped within 15-20 working days.
PSMB8 protein (His tag)
80R-2608 100 ug
EUR 322
Description: Purified recombinant PSMB8 protein
Mouse PSMB8 shRNA Plasmid
20-abx971337
  • EUR 801.00
  • EUR 1121.00
  • 150 µg
  • 300 µg
  • Shipped within 15-20 working days.
PSMB8 Recombinant Protein (Human)
RP085356 100 ug Ask for price
PSMB8 Recombinant Protein (Rat)
RP222629 100 ug Ask for price
PSMB8 Recombinant Protein (Mouse)
RP165287 100 ug Ask for price
Monoclonal PSMB8 Antibody (monoclonal) (M01), Clone: 1B3
AMM03954G 0.1mg
EUR 484
Description: A Monoclonal antibody against Human PSMB8 (monoclonal) (M01). The antibodies are raised in Mouse and are from clone 1B3. This antibody is applicable in WB, IHC and IF, IP, E
Proteasome Subunit Beta Type-8 (PSMB8) Antibody
20-abx114776
  • EUR 732.00
  • EUR 398.00
  • 150 ul
  • 50 ul
  • Shipped within 5-10 working days.
Proteasome Subunit Beta Type-8 (PSMB8) Antibody
20-abx141396
  • EUR 370.00
  • EUR 606.00
  • EUR 300.00
  • 100 ul
  • 200 ul
  • 50 ul
  • Shipped within 5-10 working days.
Proteasome Subunit Beta Type-8 (PSMB8) Antibody
abx011689-100ul 100 ul
EUR 411
  • Shipped within 5-10 working days.
Proteasome Subunit Beta Type-8 (PSMB8) Antibody
20-abx006453
  • EUR 411.00
  • EUR 592.00
  • EUR 182.00
  • EUR 314.00
  • 100 ul
  • 200 ul
  • 20 ul
  • 50 ul
  • Shipped within 5-10 working days.
Proteasome Subunit Beta Type-8 (PSMB8) Antibody
abx028154-400ul 400 ul
EUR 523
  • Shipped within 5-10 working days.
Proteasome Subunit Beta Type-8 (PSMB8) Antibody
abx028154-80l 80 µl
EUR 286
  • Shipped within 5-10 working days.
Proteasome subunit beta type-8 (PSMB8) Antibody
20-abx211178
  • EUR 411.00
  • EUR 300.00
  • 100 ul
  • 50 ul
  • Shipped within 5-10 working days.
Proteasome subunit beta type-8 (PSMB8) Antibody
20-abx211851
  • EUR 411.00
  • EUR 300.00
  • 100 ul
  • 50 ul
  • Shipped within 5-10 working days.
[KO Validated] PSMB8 Rabbit pAb
A19933-100ul 100 ul
EUR 410
[KO Validated] PSMB8 Rabbit pAb
A19933-200ul 200 ul
EUR 571
[KO Validated] PSMB8 Rabbit pAb
A19933-20ul 20 ul
EUR 221
[KO Validated] PSMB8 Rabbit pAb
A19933-50ul 50 ul
EUR 287
PSMB8 ORF Vector (Human) (pORF)
ORF028453 1.0 ug DNA
EUR 405
Psmb8 ORF Vector (Rat) (pORF)
ORF074211 1.0 ug DNA
EUR 506
Psmb8 ORF Vector (Mouse) (pORF)
ORF055097 1.0 ug DNA
EUR 506
PSMB8 ELISA Kit (Human) (OKCD02695)
OKCD02695 96 Wells
EUR 792
Description: Description of target: The proteasome is a multicatalytic proteinase complex which is characterized by its ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH. The proteasome has an ATP-dependent proteolytic activity. This subunit is involved in antigen processing to generate class I binding peptides. Replacement of PSMB5 by PSMB8 increases the capacity of the immunoproteasome to cleave model peptides after hydrophobic and basic residues. Acts as a major component of interferon gamma-induced sensitivity. Plays a key role in apoptosis via the degradation of the apoptotic inhibitor MCL1. May be involved in the inflammatory response pathway. In cancer cells, substitution of isoform 1 (E2) by isoform 2 (E1) results in immunoproteasome deficiency. Required for the differentiation of preadipocytes into adipocytes.;Species reactivity: Human;Application: ;Assay info: Assay Methodology: Quantitative Sandwich ELISA;Sensitivity: 0.117 ng/mL
PSMB8 ELISA Kit (Dog) (OKEH03975)
OKEH03975 96 Wells
EUR 844
Description: Description of target: ;Species reactivity: Dog;Application: ;Assay info: Assay Methodology: Quantitative Sandwich ELISA;Sensitivity: 0.064 ng/mL
PSMB8 ELISA Kit (Human) (OKEH07770)
OKEH07770 96 Wells
EUR 1092
Description: Description of target: The proteasome is a multicatalytic proteinase complex with a highly ordered ring-shaped 20S core structure. The core structure is composed of 4 rings of 28 non-identical subunits; 2 rings are composed of 7 alpha subunits and 2 rings are composed of 7 beta subunits. Proteasomes are distributed throughout eukaryotic cells at a high concentration and cleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway. An essential function of a modified proteasome, the immunoproteasome, is the processing of class I MHC peptides. This gene encodes a member of the proteasome B-type family, also known as the T1B family, that is a 20S core beta subunit. This gene is located in the class II region of the MHC (major histocompatibility complex). Expression of this gene is induced by gamma interferon and this gene product replaces catalytic subunit 3 (proteasome beta 5 subunit) in the immunoproteasome. Proteolytic processing is required to generate a mature subunit. Two alternative transcripts encoding two isoforms have been identified; both isoforms are processed to yield the same mature subunit.;Species reactivity: Human;Application: ELISA;Assay info: Assay Methodology: Quantitative Sandwich ELISA;Sensitivity: 0.06ng/mL
PSMB8 sgRNA CRISPR Lentivector set (Human)
K1740301 3 x 1.0 ug
EUR 339
Psmb8 sgRNA CRISPR Lentivector set (Mouse)
K4425601 3 x 1.0 ug
EUR 339
Psmb8 sgRNA CRISPR Lentivector set (Rat)
K6921501 3 x 1.0 ug
EUR 339
PSMB8 sgRNA CRISPR Lentivector (Human) (Target 1)
K1740302 1.0 ug DNA
EUR 154
PSMB8 sgRNA CRISPR Lentivector (Human) (Target 2)
K1740303 1.0 ug DNA
EUR 154
PSMB8 sgRNA CRISPR Lentivector (Human) (Target 3)
K1740304 1.0 ug DNA
EUR 154
Psmb8 sgRNA CRISPR Lentivector (Mouse) (Target 1)
K4425602 1.0 ug DNA
EUR 154
Psmb8 sgRNA CRISPR Lentivector (Mouse) (Target 2)
K4425603 1.0 ug DNA
EUR 154
Psmb8 sgRNA CRISPR Lentivector (Mouse) (Target 3)
K4425604 1.0 ug DNA
EUR 154
Human Proteasome subunit beta type-8 (PSMB8)
1-CSB-EP018886HU
  • EUR 380.00
  • EUR 214.00
  • EUR 1309.00
  • EUR 560.00
  • EUR 873.00
  • EUR 262.00
  • 100ug
  • 10ug
  • 1MG
  • 200ug
  • 500ug
  • 50ug
  • MW: 42.7 kDa
  • Buffer composition: Tris-based buffer with 50% glycerol.
Description: Recombinant Human Proteasome subunit beta type-8(PSMB8) expressed in E.coli
Psmb8 sgRNA CRISPR Lentivector (Rat) (Target 1)
K6921502 1.0 ug DNA
EUR 154
Psmb8 sgRNA CRISPR Lentivector (Rat) (Target 2)
K6921503 1.0 ug DNA
EUR 154
Psmb8 sgRNA CRISPR Lentivector (Rat) (Target 3)
K6921504 1.0 ug DNA
EUR 154
PSMB8 Protein Vector (Human) (pPB-C-His)
PV113810 500 ng
EUR 552
PSMB8 Protein Vector (Human) (pPB-N-His)
PV113811 500 ng
EUR 552
PSMB8 Protein Vector (Human) (pPM-C-HA)
PV113812 500 ng
EUR 552
PSMB8 Protein Vector (Human) (pPM-C-His)
PV113813 500 ng
EUR 552
Recombinant Human PSMB8 Protein, His, E.coli-1mg
QP13190-1mg 1mg
EUR 2757
Recombinant Human PSMB8 Protein, His, E.coli-20ug
QP13190-20ug 20ug
EUR 201
Recombinant Human PSMB8 Protein, His, E.coli-5ug
QP13190-5ug 5ug
EUR 155
PSMB8 Protein Vector (Rat) (pPB-C-His)
PV296842 500 ng
EUR 603
PSMB8 Protein Vector (Rat) (pPB-N-His)
PV296843 500 ng
EUR 603
PSMB8 Protein Vector (Rat) (pPM-C-HA)
PV296844 500 ng
EUR 603
PSMB8 Protein Vector (Rat) (pPM-C-His)
PV296845 500 ng
EUR 603
PSMB8 Protein Vector (Mouse) (pPB-C-His)
PV220386 500 ng
EUR 603
PSMB8 Protein Vector (Mouse) (pPB-N-His)
PV220387 500 ng
EUR 603
PSMB8 Protein Vector (Mouse) (pPM-C-HA)
PV220388 500 ng
EUR 603
PSMB8 Protein Vector (Mouse) (pPM-C-His)
PV220389 500 ng
EUR 603
Psmb8 3'UTR GFP Stable Cell Line
TU167173 1.0 ml Ask for price
PSMB8 3'UTR Luciferase Stable Cell Line
TU019046 1.0 ml
EUR 1394
Psmb8 3'UTR Luciferase Stable Cell Line
TU117173 1.0 ml Ask for price
PSMB8 3'UTR GFP Stable Cell Line
TU069046 1.0 ml
EUR 1394
Psmb8 3'UTR GFP Stable Cell Line
TU266964 1.0 ml Ask for price
Psmb8 3'UTR Luciferase Stable Cell Line
TU216964 1.0 ml Ask for price
PSMB8 Lentiviral Vector (Rat) (CMV) (pLenti-GIII-CMV)
LV700471 1.0 ug DNA
EUR 514
PSMB8 Lentiviral Vector (Rat) (UbC) (pLenti-GIII-UbC)
LV700475 1.0 ug DNA
EUR 514
PSMB8 Lentiviral Vector (Rat) (EF1a) (pLenti-GIII-EF1a)
LV700476 1.0 ug DNA
EUR 514
Monkey Low Molecular Mass Protein 7 (PSMB8) ELISA Kit
abx360252-96tests 96 tests
EUR 825
  • Shipped within 5-12 working days.
Pig Low Molecular Mass Protein 7 (PSMB8) ELISA Kit
abx361997-96tests 96 tests
EUR 825
  • Shipped within 5-12 working days.
Rabbit Low Molecular Mass Protein 7 (PSMB8) ELISA Kit
abx362317-96tests 96 tests
EUR 825
  • Shipped within 5-12 working days.
Cow Low Molecular Mass Protein 7 (PSMB8) ELISA Kit
abx515621-96tests 96 tests
EUR 911
  • Shipped within 5-12 working days.
Dog Low Molecular Mass Protein 7 (PSMB8) ELISA Kit
abx515622-96tests 96 tests
EUR 911
  • Shipped within 5-12 working days.
Human Low Molecular Mass Protein 7 (PSMB8) ELISA Kit
abx515623-96tests 96 tests
EUR 739
  • Shipped within 5-12 working days.
Mouse Low Molecular Mass Protein 7 (PSMB8) ELISA Kit
abx515624-96tests 96 tests
EUR 739
  • Shipped within 5-12 working days.
Pig Low Molecular Mass Protein 7 (PSMB8) ELISA Kit
abx515625-96tests 96 tests
EUR 911
  • Shipped within 5-12 working days.
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