The Ct serovars E (DSM 19131) and L2 (DSM 19102) used in this study were propagated either in HeLa human epithelial cells (ATCC® CCL-2TM) or McCoy [McCoyB] mouse fibroblasts (ATCC® CRL-1696TM) with modifications, as previously described [24 ,25 (link)].
Ccl 2tm
Manufactured by American Type Culture Collection
Sourced in United States
The CCL-2TM is a laboratory equipment product offered by American Type Culture Collection. It is designed for the cultivation and maintenance of cell cultures. The core function of the CCL-2TM is to provide a controlled environment for the growth and propagation of various cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Hela cell, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Htb 85, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by American Type Culture Collection (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Htb 26tm, by American Type Culture Collection (1 mentions)
Crl 1469, by American Type Culture Collection (1 mentions)
8 protocols using ccl 2tm
1
Propagation of Chlamydia Serovars in Cell Lines
2
3D Cell Culture Model Characterization
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This 3D model was first implemented using HeLa human cervical adenocarcinoma cells (ATCC® CCL-2TM) and most of the experiments were performed with this immortalized cell line. For comparison, primary dermal fibroblasts (ATCC® PCS-201-010TM) (human fibroblasts derived from the foreskin of male African newborn with spindle-shaped morphology) were used for 3D-CCM characterization and evaluation of the reproducibility. All the cells were purchased in 2016 from ATCC© (ATCC France, Molsheim, France). Several ampoules containing cells at early passage were then generated and frozen to have a stock. HeLa cells were used until passage 25 and fibroblasts until passage 15 before returning to stock.
3
Quantifying Intracellular Salmonella Infection
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GFP-expressing S. enterica serovar Hadar or non-fluorescent S. enterica serovar Hadar were treated with 500 μg/mL of Abi-Se07 and incubated for 30′ at 37°C. Then, confluent HeLa cells (ATCC® CCL-2TM) or QM7 cells (ATCC® CRL-1962TM) were infected at a multiplicity of infection (MOI) of 50 with GFP expressing S. enterica serovar Hadar, or S. enterica serovar Hadar, respectively. To quantify cell-associated Salmonella in HeLa cells, fluorescence images were taken with Cytation 5 and fluorescent bacteria quantified. To measure intracellular bacteria, HeLa or QM7 cells were then treated with 100 μg/mL gentamicin and incubated for 1 h at 37°C. Gentamicin is a non-membrane permeable antibiotic and will kill non-internalized bacteria. Finally, cells were washed 3× with PBS. For HeLa cells, fluorescence images were taken with Cytation 5 and fluorescent bacteria quantified. For QM7 cells, 0.1% saponin solution was added and allowed to incubate with cells for 10′ at 37°C. The mixture was then vortexed and homogenized for cell lysis. Lysates were serially diluted and plated on LB agar for CFU counting. Statistical analysis was performed using unpaired student t-test.
4
HeLa Cell Culture Protocol
5
Genetic Variant Linkage Analysis in Human Osteoblasts
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The Haploview software [36] (link) was used to calculate and represent the degree of linkage disequilibrium between the genotyped common variants using the default parameters.
A C C E P T E D M A N U S C R I P T FBS, 1% penicillin/streptomycin and 1x Glutamax (Gibco, Life Technologies), at 37ºC and 5% of CO 2 . Human primary osteoblasts (hOB) were used for eQTL assays. They were obtained from trabecular bone of women who underwent knee replacement due to osteoarthritis and who did not have any other pathology that could affect the bone status. Bony tissue was cut up into small pieces, washed in phosphate buffered saline (PBS; Gibco, Life technologies) to remove non-adherent cells, and placed on a 140 mm culture plate. Samples were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 0.4% fungizone (Gibco, Life Technologies) and 100 g/ml ascorbic acid (Sigma-Aldrich). DNA and RNA extractions were performed at maximum passage 2. HeLa and HEK293 cell lines were obtained from ATCC (ATCC ® HTB-85 and ATCC ® CCL-2 TM , respectively) and grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37ºC and 5% CO 2 .
A C C E P T E D M A N U S C R I P T FBS, 1% penicillin/streptomycin and 1x Glutamax (Gibco, Life Technologies), at 37ºC and 5% of CO 2 . Human primary osteoblasts (hOB) were used for eQTL assays. They were obtained from trabecular bone of women who underwent knee replacement due to osteoarthritis and who did not have any other pathology that could affect the bone status. Bony tissue was cut up into small pieces, washed in phosphate buffered saline (PBS; Gibco, Life technologies) to remove non-adherent cells, and placed on a 140 mm culture plate. Samples were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 0.4% fungizone (Gibco, Life Technologies) and 100 g/ml ascorbic acid (Sigma-Aldrich). DNA and RNA extractions were performed at maximum passage 2. HeLa and HEK293 cell lines were obtained from ATCC (ATCC ® HTB-85 and ATCC ® CCL-2 TM , respectively) and grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37ºC and 5% CO 2 .
6
Fluorescent Nanoparticle Labeling of HeLa Cells
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40 nm yellow/green and 100 nm red fluorescently labelled carboxylated polystyrene nanoparticles were purchased from ThermoFisher (Eugene, OR, USA) and were used without further chemical modification. HeLa cells were acquired from American Type Culture Collection (ATTC; Manassas, VA, USA; CCL-2TM, lot no. 61647128). Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Life Technologies, Eugene, OR, USA), Dulbecco’s Phosphate Buffered Saline (PBS; Gibco, Life Technologies, Eugene, OR, USA), foetal bovine serum (FBS; Gibco, Life Technologies, Eugene, OR, USA) and Trypsin-EDTA (Gibco, Life Technologies, Eugene, OR, USA) were purchased from ThermoFisher (Eugene, OR, USA).
7
Culturing MDA-MB-231, Panc-1, and HeLa Cells
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MDA-MB-231, Panc-1 and HeLa cells were purchased from the American Type Culture Collection (ATCC®) (VA) (HTB-26 TM, CRL-1469TM and CCL-2 TM respectively). Cells were cultured in low-glucose Dulbecco's Modified Medium (DMEM) (Merck, Germany, D6046) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, 10270). MDA-MB-231 cells were cultured at 5% CO2 concentration and 37 °C and Panc-1 and HeLa were cultured at 10% CO2 and 37 °C.
8
HeLa Cell Culture and Knockout Variants
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Wild type HeLa cells (CCL2TM from American Type Culture Collection, Rockville, MD, United States) were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 4.5 g/L of D -glucose, and glutaMAX (61965-026, GIBCO, ThermoFisher Scientific, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (16000-044, GIBCO, ThermoFisher Scientific, Switzerland), 1% penicillin-streptomycin (15070-063, GIBCO, ThermoFisher Scientific, Switzerland), and maintained under standard conditions (5% CO2, 95% O2, 37°C). β-arrestin1 KO, β-arrestin2 KO, and β-arrestin1/2 KO HeLa cells, were cultured in the same medium and under standard culture conditions as described for wt cells. Cells were detached using 10 mM ethylenediaminetetraacetic acid (EDTA)/PBS for 3 min under standard culture conditions.
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