Dear Ruiying,

The followings list some cell lines used by different researchers for the same purpose of yours.

1-Identification of Megalin/gp330 as a Receptor for Lipoprotein(a) In Vitro (see attached file).

Andreas Niemeier, Thomas Willnow, Hans Dieplinger, Christian Jacobsen, Nicolette Meyer,

Jan Hilpert, Ulrike Beisiegel

Methods

Generation of Yolk Sac Cell Lines

To generate cell lines 10A and 1461, individual embryos from

matings of mice heterozygous for the LRP gene defect (LRP6) were

isolated at day 10.5 of gestation, and the embryonic membranes were

removed. The yolk sacs were placed in ice-cold, 0.05% trypsinEDTA

solution and kept overnight at 4°C. On the following day, the

samples were incubated at 37°C and disaggregated by vigorous

pipetting. The cell suspension was plated on a 60-mm culture dish

and grown to confluence. Individual cell clones were genotyped by

Southern blot analysis to identify those heterozygous (10A) or

homozygous (1461) for the LRP gene defect. To obtain cell line 6A3,

mice doubly homozygous for the LRP and the megalin gene defect

(LRP2/2, megalin2/2) were produced by mating of LRP6 and

megalin6 mice. Embryos from this mating were isolated at day 10.5

of gestation, and individual yolk sac cell clones were isolated.

Because we were unable to obtain doubly homozygous–deficient

cell lines, we treated line 6A (LRP6, megalin2/2) with Pseudomonas

aeruginosa exotoxin A, a ligand for the LRP that can be used

as a negative selectable marker against cells expressing LRP.52

Accordingly, Pseudomonas aeruginosa exotoxin A–resistant cell

clones were isolated from a pool of 6A cells, and the doubly deficient

(LRP2/2, megalin2/2) line 6A3 was identified by Southern blot

analysis. To immortalize all yolk sac lines, they were transfected

with a plasmid encoding Simian virus 40 large T antigen.

Cell Culture and Protein Preparation

Cells were cultured in Dulbecco’s modified Eagle’s medium

(DMEM) without glutamine and with 10% FCS. For protein blotting

procedures, cells were solubilized in 1% Triton X-100, 50 mmol/L

Tris, 2 mmol/L CaCl2, and 80 mmol/L NaCl, pH 8.0. A mix of

protease inhibitors (Calbiochem) was added, including 1 mmol/L

pepstatin A, 10 mmol/L chymostatin, 10 mmol/L leupeptin, and

10 mmol/L antipain. Protein concentrations were determined by

standard procedures according to Lowry et al.53

Gel Electrophoresis and Antibodies

SDS–polyacrylamide gel electrophoresis (PAGE) was performed

according to Neville.54 For 125I-labeled proteins, gels were dried and

exposed to an autoradiography film (Cronex, DuPont). Nonlabeled

proteins were electroblotted to nitrocellulose for nonspecific protein

stain (Ponceau S solution, Serva) and immunodetection with specific

antibodies. Polyclonal antibodies against megalin/gp330, LRP, and

the LDLR were given to us by J. Herz (University of Texas

Southwestern Medical Center, Dallas, Tex), and the polyclonal

anti-VLDLR was kindly provided by M. Gåfvels (Karolinska Institute,

Huddinge, Sweden). Peroxidase-labeled goat anti-rabbit antibodies

(Dianova) were used with chloronaphthol as the substrate for

secondary antibodies. Southern blot analyses were performed according

to standard procedures.55

2-Multidrug Resistance and the Lung Resistance-Related Protein in Human Colon Carcinoma SW-620 Cells (see attached file).

Masaki Kitazono, Tomoyuki Sumizawa, Yuji Takebayashi, Zhe-Sheng Chen,

Tatsuhiko Furukawa, Shuichi Nagayama, Ayako Tani, Sonshin Takao, Takashi

Aikou, Shin-ichi Akiyama

Cell culture. SW-620, a human colon carcinoma cell line, was obtained from

A. T. Fojo (National Cancer Institute, Bethesda, MD). Cells were grown at 37 °C

in 5% CO2–95% air in RPMI-1640 medium containing 10% newborn calf serum,

2 mM glutamine, and 100 U/mL penicillin. Sodium butyrate was added to

a final concentration of 2 mM to induce differentiation. Culture medium alone or

containing sodium butyrate was changed daily. KB-3-1 cells were cultured as

described (15). Multidrug-resistant KB-C2 cells, overexpressing MDR1 messenger

RNA (mRNA) and P-gp, and C-A120 cells, a non-P-gp-mediated multidrugresistant

cell line overexpressing MRP, were isolated from KB-3-1 cell cultures

and maintained as described (15,16). HT-1080 fibrosarcoma cells were used as

positive controls for LRP.

3-Molecular and Cellular Characterization of Imexon-resistant RPMI8226/I Myeloma Cells1 (see attached file).

Katerina Dvorakova, Claire M. Payne,

Margaret E. Tome, Margaret M. Briehl,

Miguel A. Vasquez, Caroline N. Waltmire, Amy Coon,

and Robert T. Dorr2

Cell Cultures and Viability Assays. The human myeloma

RPMI8226 cell line was obtained from the American Type

Culture Collection (Manassas, VA). RPMI8226/Dox40 and

RPMI8226/MR20 cell lines were obtained from Dr. Alan List

(University of Arizona). The imexon-resistant RPMI8266/I cell

line was established in our laboratory by continuous exposure

of RPMI8226 cells to increasing concentrations of

imexon (up to a final concentration of 90 M). RPMI8226/I

cells were then maintained in 90 M imexon for more than 6

months. All cell lines were cultured at 37°C in 5% CO2 in

RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented

with 10% (v/v) heat-inactivated bovine calf serum

(Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, 100

units/ml penicillin, and 100 g/ml streptomycin. Cellular dehydrogenase

activity, which is considered to reflect mitochondrial

activity and cell viability, was measured by a microculture

MTT assay based on the ability of living cells to

reduce MTT (Sigma Chemical Co.) to a blue formazan according

to the method of Mosmann (10).

Hoping this will be helpful,

Rafik

Dear Ruiying,

The followings list some cell lines used by different researchers for the same purpose of yours.

1-Identification of Megalin/gp330 as a Receptor for Lipoprotein(a) In Vitro (see attached file).

Andreas Niemeier, Thomas Willnow, Hans Dieplinger, Christian Jacobsen, Nicolette Meyer,

Jan Hilpert, Ulrike Beisiegel

Methods

Generation of Yolk Sac Cell Lines

To generate cell lines 10A and 1461, individual embryos from

matings of mice heterozygous for the LRP gene defect (LRP6) were

isolated at day 10.5 of gestation, and the embryonic membranes were

removed. The yolk sacs were placed in ice-cold, 0.05% trypsinEDTA

solution and kept overnight at 4°C. On the following day, the

samples were incubated at 37°C and disaggregated by vigorous

pipetting. The cell suspension was plated on a 60-mm culture dish

and grown to confluence. Individual cell clones were genotyped by

Southern blot analysis to identify those heterozygous (10A) or

homozygous (1461) for the LRP gene defect. To obtain cell line 6A3,

mice doubly homozygous for the LRP and the megalin gene defect

(LRP2/2, megalin2/2) were produced by mating of LRP6 and

megalin6 mice. Embryos from this mating were isolated at day 10.5

of gestation, and individual yolk sac cell clones were isolated.

Because we were unable to obtain doubly homozygous–deficient

cell lines, we treated line 6A (LRP6, megalin2/2) with Pseudomonas

aeruginosa exotoxin A, a ligand for the LRP that can be used

as a negative selectable marker against cells expressing LRP.52

Accordingly, Pseudomonas aeruginosa exotoxin A–resistant cell

clones were isolated from a pool of 6A cells, and the doubly deficient

(LRP2/2, megalin2/2) line 6A3 was identified by Southern blot

analysis. To immortalize all yolk sac lines, they were transfected

with a plasmid encoding Simian virus 40 large T antigen.

Cell Culture and Protein Preparation

Cells were cultured in Dulbecco’s modified Eagle’s medium

(DMEM) without glutamine and with 10% FCS. For protein blotting

procedures, cells were solubilized in 1% Triton X-100, 50 mmol/L

Tris, 2 mmol/L CaCl2, and 80 mmol/L NaCl, pH 8.0. A mix of

protease inhibitors (Calbiochem) was added, including 1 mmol/L

pepstatin A, 10 mmol/L chymostatin, 10 mmol/L leupeptin, and

10 mmol/L antipain. Protein concentrations were determined by

standard procedures according to Lowry et al.53

Gel Electrophoresis and Antibodies

SDS–polyacrylamide gel electrophoresis (PAGE) was performed

according to Neville.54 For 125I-labeled proteins, gels were dried and

exposed to an autoradiography film (Cronex, DuPont). Nonlabeled

proteins were electroblotted to nitrocellulose for nonspecific protein

stain (Ponceau S solution, Serva) and immunodetection with specific

antibodies. Polyclonal antibodies against megalin/gp330, LRP, and

the LDLR were given to us by J. Herz (University of Texas

Southwestern Medical Center, Dallas, Tex), and the polyclonal

anti-VLDLR was kindly provided by M. Gåfvels (Karolinska Institute,

Huddinge, Sweden). Peroxidase-labeled goat anti-rabbit antibodies

(Dianova) were used with chloronaphthol as the substrate for

secondary antibodies. Southern blot analyses were performed according

to standard procedures.55

2-Multidrug Resistance and the Lung Resistance-Related Protein in Human Colon Carcinoma SW-620 Cells (see attached file).

Masaki Kitazono, Tomoyuki Sumizawa, Yuji Takebayashi, Zhe-Sheng Chen,

Tatsuhiko Furukawa, Shuichi Nagayama, Ayako Tani, Sonshin Takao, Takashi

Aikou, Shin-ichi Akiyama

Cell culture. SW-620, a human colon carcinoma cell line, was obtained from

A. T. Fojo (National Cancer Institute, Bethesda, MD). Cells were grown at 37 °C

in 5% CO2–95% air in RPMI-1640 medium containing 10% newborn calf serum,

2 mM glutamine, and 100 U/mL penicillin. Sodium butyrate was added to

a final concentration of 2 mM to induce differentiation. Culture medium alone or

containing sodium butyrate was changed daily. KB-3-1 cells were cultured as

described (15). Multidrug-resistant KB-C2 cells, overexpressing MDR1 messenger

RNA (mRNA) and P-gp, and C-A120 cells, a non-P-gp-mediated multidrugresistant

cell line overexpressing MRP, were isolated from KB-3-1 cell cultures

and maintained as described (15,16). HT-1080 fibrosarcoma cells were used as

positive controls for LRP.

3-Molecular and Cellular Characterization of Imexon-resistant RPMI8226/I Myeloma Cells1 (see attached file).

Katerina Dvorakova, Claire M. Payne,

Margaret E. Tome, Margaret M. Briehl,

Miguel A. Vasquez, Caroline N. Waltmire, Amy Coon,

and Robert T. Dorr2

Cell Cultures and Viability Assays. The human myeloma

RPMI8226 cell line was obtained from the American Type

Culture Collection (Manassas, VA). RPMI8226/Dox40 and

RPMI8226/MR20 cell lines were obtained from Dr. Alan List

(University of Arizona). The imexon-resistant RPMI8266/I cell

line was established in our laboratory by continuous exposure

of RPMI8226 cells to increasing concentrations of

imexon (up to a final concentration of 90 M). RPMI8226/I

cells were then maintained in 90 M imexon for more than 6

months. All cell lines were cultured at 37°C in 5% CO2 in

RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented

with 10% (v/v) heat-inactivated bovine calf serum

(Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, 100

units/ml penicillin, and 100 g/ml streptomycin. Cellular dehydrogenase

activity, which is considered to reflect mitochondrial

activity and cell viability, was measured by a microculture

MTT assay based on the ability of living cells to

reduce MTT (Sigma Chemical Co.) to a blue formazan according

to the method of Mosmann (10).

Hoping this will be helpful,

Rafik

Mouse embryonic fibroblasts (MEF) cells as well as CHO K1 (Chinese Hamster Ovary cells) are cell lines with high LRP-1 expression. For both cell lines there are corresponding LRP-1-ko cells (as negative control) available. Let me know if I can be of any further help.

Best,

Steffen

See also:

https://www.researchgate.net/publication/285215509_Endothelial_LRP1_transports_amyloid-b1-42_across_the_blood-brain_barrier

Article Endothelial LRP1 transports amyloid-β1–42 across the blood-b...

Hello

CHO-K1, CHO 13-5-1  , MEF, and primary human skin fibroblast cells lines

http://www.jbc.org/content/285/30/23047.full

Sorry, but there is probably a typo in the last comment by Saleh Alkarim: CHO 13-5-1 do NOT express LRP-1!! These are LRP1-ko cells!

Thank you so much for all your help. it's really helpful!