Leukemic Stem Cell Culture in Cytokine-Free Medium

Leukemia-initiating cells, also known as leukemic stem cells (LSCs), are experimentally defined by their ability to engraft immunocompromised mice and are believed to be a major cause of relapse in acute myeloid leukemia (AML). Despite the aggressive characteristics of acute leukemia, AML blasts are difficult to culture once removed from the patient, and LSCs, which are a minor fraction of the blast population, are especially difficult to transplant after culture. This impedes development of new therapies for AML that target LSCs. Here, we present a simple strategy to culture LSCs in cytokine-free medium and to perform flow cytometric analysis of the resulting cell population for the characterization of LSCs maintenance and differentiation in Gentaur.

Adenosine A ₁ and A _2a receptors modulate the nitrergic system in cell culture from dorsomedial medulla oblongata

Adenosine and nitric oxide act on the fine-tuning regulation of neural cardiovascular control in the nucleus tractus solitarius (NTS). Although the interaction between adenosine and NO is well known in the periphery, the mechanisms by which adenosine interferes in the dynamics of nitrergic neurotransmission, related to neural control of circulation, are not completely understood and might be relevant for individuals predisposed to hypertension. In this study we evaluate the interaction between adenosinergic and nitrergic systems in cell culture from the dorsomedial medulla oblongata of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR).

Using quantification of nitrite levels, RT-PCR analysis and RNA interference we demonstrate that adenosine A₁ (A₁R) and A_2a receptor (A_2aR) agonists induce a concentration-dependent decrease and increase of nitrite and nNOS mRNA levels in cultured cells from WKY and SHR, respectively.

These effects in nitrite levels are attenuated by the administration of A₁R and A_2aR selective antagonists, CPT and ZM 241385. Furthermore, knockdown of A₁R and A_2aR show an increase and decrease of nNOS mRNA levels, respectively. Pretreatment with the nonselective inhibitor of NOS, L-NAME, abolishes nitrite-increased levels triggered by CGS 21680 in WKY and SHR cells.

Finally, it is shown that the cAMP-PKA pathway is involved in A₁R and A_2aR-mediated decrease and increase in nitrite levels in SHR and WKY cells. Our results highlight the influence of adenosine on nitric oxide levels in cultured cells from dorsal medulla oblongata of neonate WKY and SHR rats. In part, the modulatory profile is different in the SHR strain, que es la parafina.

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Control of IgG glycosylation in CHO cell perfusion cultures by GReBA mathematical model supported by a novel targeted feed, TAFE

The N-linked glycosylation pattern is an important quality attribute of therapeutic glycoproteins. It has been reported by our group and by others that different carbon sources, such as glucose, mannose and galactose, can differently impact the glycosylation profile of glycoproteins in mammalian cell culture. Acting on the sugar feeding is thus an attractive strategy to tune the glycan pattern. However, in case of feeding of more than one carbon source simultaneously, the cells give priority to the one with the highest uptake rate, which limits the usage of this tuning, e.g. the cells favor consuming glucose in comparison to galactose. We present here a new feeding strategy (named ‘TAFE’ for targeted feeding) for perfusion culture to adjust the concentrations of fed sugars influencing the glycosylation. The strategy consists in setting the sugar feeding such that the cells are forced to consume these substrates at a target cell specific consumption rate decided by the operator and taking into account the cell specific perfusion rate (CSPR).

eaaci2015

DAPI (Molecular Biology Grade)
CE118	GeneOn	25 mg	EUR 159.6

DAPI (Molecular Biology Grade)
CE119	GeneOn	100 mg	EUR 382.8

Dimethylsulfoxide (Molecular Biology Grade)
CE120	GeneOn	100 ml	EUR 66

Dimethylsulfoxide (Molecular Biology Grade)
CE121	GeneOn	500 ml	EUR 110.4

DTT (Molecular Biology Grade)
CE131	GeneOn	5 g	EUR 93.6

DTT (Molecular Biology Grade)
CE132	GeneOn	10 g	EUR 133.2

DTT (Molecular Biology Grade)
CE133	GeneOn	25 g	EUR 243.6

Glycine (Molecular Biology Grade)
CE158	GeneOn	1 kg	EUR 84

Glycine (Molecular Biology Grade)
CE159	GeneOn	5 kg	EUR 228

HEPES (Molecular Biology Grade)
CE171	GeneOn	100 g	EUR 98.4

HEPES (Molecular Biology Grade)
CE172	GeneOn	500 g	EUR 268.8

HEPES (Molecular Biology Grade)
CE173	GeneOn	1 kg	EUR 424.8

Lysozyme (Molecular Biology Grade)
CE188	GeneOn	1 g	EUR 70.8

Lysozyme (Molecular Biology Grade)
CE189	GeneOn	10 g	EUR 247.2

NAD (Molecular Biology Grade)
CE196	GeneOn	1 g	EUR 72

NAD (Molecular Biology Grade)
CE197	GeneOn	5 g	EUR 165.6

NBT (Molecular Biology Grade)
CE209	GeneOn	1 g	EUR 123.6

NBT (Molecular Biology Grade)
CE210	GeneOn	5 g	EUR 360

Tris (Molecular Biology Grade)
CE237	GeneOn	500 g	EUR 106.8

Tris (Molecular Biology Grade)
CE238	GeneOn	1 kg	EUR 153.6

Tris (Molecular Biology Grade)
CE239	GeneOn	5 kg	EUR 535.2

Tween20 (Molecular Biology Grade)
CE242	GeneOn	1 l	EUR 106.8

Water (Molecular Biology Grade)
CE243	GeneOn	500 ml	EUR 62.4

Water (Molecular Biology Grade)
CE244	GeneOn	1 l	EUR 67.2

100mL Molecular Biology Grade
46-000-CI	Scientific Laboratory Supplies	PK6	EUR 70.68

500mL Molecular Biology Grade
46-000-CV	Scientific Laboratory Supplies	PK6	EUR 124.26

Ammonium sulfate (Molecular Biology Grade)
CE105	GeneOn	250 g	EUR 55.2

Ammonium sulfate (Molecular Biology Grade)
CE106	GeneOn	1 kg	EUR 72

Ammonium sulfate (Molecular Biology Grade)
CE107	GeneOn	5 kg	EUR 153.6

Bis-Acrylamid (Molecular Biology Grade)
CE110	GeneOn	50 g	EUR 94.8

Bis-Acrylamid (Molecular Biology Grade)
CE111	GeneOn	250 g	EUR 259.2

Formamide deionized (Molecular Biology Grade)
CE145	GeneOn	500 ml	EUR 87.6

Formamide deionized (Molecular Biology Grade)
CE146	GeneOn	1 l	EUR 120

Glycerol 87 % (Molecular Biology Grade)
CE154	GeneOn	1 l	EUR 93.6

Glycerol waterfree (Molecular Biology Grade)
CE155	GeneOn	500 ml	EUR 78

Glycerol waterfree (Molecular Biology Grade)
CE156	GeneOn	1 l	EUR 102

Glycerol waterfree (Molecular Biology Grade)
CE157	GeneOn	2.5 l	EUR 170.4

Guanidine - Hydrochloride (Molecular Biology Grade)
CE160	GeneOn	100 g	EUR 93.6

Guanidine - Hydrochloride (Molecular Biology Grade)
CE161	GeneOn	250 g	EUR 153.6

Guanidine - Hydrochloride (Molecular Biology Grade)
CE162	GeneOn	500 g	EUR 232.8

Guanidine - Hydrochloride (Molecular Biology Grade)
CE163	GeneOn	1 kg	EUR 352.8

Guanidine Thiocyanate (Molecular Biology Grade)
CE164	GeneOn	100 g	EUR 86.4

Guanidine Thiocyanate (Molecular Biology Grade)
CE165	GeneOn	500 g	EUR 192

Guanidine Thiocyanate (Molecular Biology Grade)
CE166	GeneOn	1 kg	EUR 307.2

Urea Crystalline (Molecular Biology Grade)
CE167	GeneOn	1 kg	EUR 72

Urea Crystalline (Molecular Biology Grade)
CE168	GeneOn	5 kg	EUR 181.2

MOPS buffer (Molecular Biology Grade)
CE194	GeneOn	100 g	EUR 102

MOPS buffer (Molecular Biology Grade)
CE195	GeneOn	250 g	EUR 169.2

Sodium chloride (Molecular Biology Grade)
CE205	GeneOn	500 g	EUR 62.4

Sodium chloride (Molecular Biology Grade)
CE206	GeneOn	1 kg	EUR 70.8

Sodium chloride (Molecular Biology Grade)
CE207	GeneOn	5 kg	EUR 123.6

D(+)-Sucrose (Molecular Biology Grade)
CE224	GeneOn	500 g	EUR 67.2

D(+)-Sucrose (Molecular Biology Grade)
CE225	GeneOn	1 kg	EUR 84

D(+)-Sucrose (Molecular Biology Grade)
CE226	GeneOn	5 kg	EUR 207.6

Tris - Hydrochloride (Molecular Biology Grade)
CE234	GeneOn	250 g	EUR 99.6

Tris - Hydrochloride (Molecular Biology Grade)
CE235	GeneOn	500 g	EUR 144

Tris - Hydrochloride (Molecular Biology Grade)
CE236	GeneOn	1 kg	EUR 223.2

TritonX-100 (Molecular Biology Grade)
CE240	GeneOn	500 ml	EUR 67.2

TritonX-100 (Molecular Biology Grade)
CE241	GeneOn	1 l	EUR 79.2

Water, Ultrapure Molecular Biology Grade
41024-4L	Biotium	4L	EUR 145.2
Description: Minimum order quantity: 1 unit of 4L

Urea, suitable for molecular biology
GE1210-1KG	Glentham Life Sciences	1 kg	EUR 106.8

Urea, suitable for molecular biology
GE1210-500G	Glentham Life Sciences	500 g	EUR 76.8

Tween 20, Molecular Biology Grade
T9100-010	GenDepot	100ml	EUR 86.4

Tween 20, Molecular Biology Grade
T9100-050	GenDepot	500ml	EUR 133.2

Tween 20, Molecular Biology Grade
T9100-100	GenDepot	1L	EUR 160.8

1L Molecular Biology Grade Water
46-000-CM	Scientific Laboratory Supplies	PK6	EUR 177.84

EDTA - Dinatriumsalz - Dihydrat (Molecular Biology Grade)
CE135	GeneOn	250 g	EUR 72

EDTA - Dinatriumsalz - Dihydrat (Molecular Biology Grade)
CE136	GeneOn	500 g	EUR 86.4

EDTA - Dinatriumsalz - Dihydrat (Molecular Biology Grade)
CE137	GeneOn	1 kg	EUR 124.8

EDTA - Dinatriumsalz - Dihydrat (Molecular Biology Grade)
CE138	GeneOn	5 kg	EUR 418.8

D(+)-Glucose waterfree (Molecular Biology Grade)
CE148	GeneOn	500 g	EUR 67.2

D(+)-Glucose waterfree (Molecular Biology Grade)
CE149	GeneOn	1 kg	EUR 75.6

D(+)-Glucose waterfree (Molecular Biology Grade)
CE150	GeneOn	5 kg	EUR 180

Yeast extract powder (Molecular Biology Grade)
CE169	GeneOn	500 g	EUR 133.2

Hyaluronidase Grade I (Molecular Biology Grade)
CE174	GeneOn	1 g	EUR 232.8

Hyaluronidase Grade I (Molecular Biology Grade)
CE175	GeneOn	5 g	EUR 920.4

Magnesium acetate - Tetrahydrate (Molecular Biology Grade)
CE190	GeneOn	500 g	EUR 98.4

NADH - Disodium salt (Molecular Biology Grade)
CE198	GeneOn	1 g	EUR 91.2

NADH - Disodium salt (Molecular Biology Grade)
CE199	GeneOn	5 g	EUR 244.8

NADP - sodium salt (Molecular Biology Grade)
CE200	GeneOn	250 mg	EUR 92.4

NADP - sodium salt (Molecular Biology Grade)
CE201	GeneOn	1 g	EUR 190.8

NADPH - Tetrasodium salt (Molecular Biology Grade)
CE202	GeneOn	25 mg	EUR 70.8

NADPH - Tetrasodium salt (Molecular Biology Grade)
CE203	GeneOn	100 mg	EUR 126

NADPH - Tetrasodium salt (Molecular Biology Grade)
CE204	GeneOn	500 mg	EUR 374.4

SSC Buffer (20X) (Molecular Biology Grade)
CE229	GeneOn	1 l	EUR 86.4

XTT sodium salt (Molecular Biology Grade)
CE250	GeneOn	100 mg	EUR 208.8

XTT sodium salt (Molecular Biology Grade)
CE251	GeneOn	500 mg	EUR 612

DiscoveryProbe? Cancer Biology-related Compounds Panel
L1003-5	ApexBio	5 mg/well	EUR 4736.4
Description: A wide range of well-characterized bioactive molecules that covers various targets related to cancer biology, including p53, EGFR and PKC etc. Facilitate your research towards the insights of gene regulation, tumorgenesis and immunotherapy etc.

Sucrose, GlenBiol, suitable for molecular biology
GC3201-1KG	Glentham Life Sciences	1 kg	EUR 90

Dendritic Cells/B Cells Antibody
abx412775-02mg	Abbexa	0.2 mg	EUR 678

Albumin fraction V (pH7,0) (Molecular Biology Grade)
CE100	GeneOn	50 g	EUR 128.4

Albumin fraction V (pH7,0) (Molecular Biology Grade)
CE101	GeneOn	100 g	EUR 193.2

Albumin fraction V (pH7,0) (Molecular Biology Grade)
CE102	GeneOn	250 g	EUR 387.6

Albumin fraction V (pH7,0) (Molecular Biology Grade)
CE103	GeneOn	500 g	EUR 656.4

Albumin fraction V (pH7,0) (Molecular Biology Grade)
CE104	GeneOn	1 kg	EUR 1162.8

Water, Ultra Pure, Sterile Molecular Biology Grade
18-193	Genesee Scientific	100ml/Unit	EUR 268

Water, Ultra Pure, Sterile Molecular Biology Grade
18-194	Genesee Scientific	500ml/Unit	EUR 294

This strategy is applied in perfusion cultures of Chinese hamster ovary (CHO) cells, illustrated by ten different regimes of sugar feeding, including glucose, galactose and mannose. Applying the TAFE strategy, different glycan profiles were obtained using the different feeding regimes. Furthermore, we successfully forced the cells to consume higher proportions of non-glucose sugars, which have lower transport rates than glucose in presence of this latter, in a controlled way.

In previous work, a mathematical model named Glycan Residues Balance Analysis (GReBA) was developed to model the glycosylation profile based on the fed carbon sources. The present data were applied to the GReBA to design a feeding regime targeting a given glycosylation profile. The ability of the model to achieve this objective was confirmed by a multi-round of leave-one-out cross-validation (LOOCV), leading to the conclusion that the GReBA model can be used to design the feeding regime of a perfusion cell culture to obtain a desired glycosylation profile.

Flow-Cytometric Method for Viability Analysis of Mycoplasma gallisepticum and Other Cell–Culture-Contaminant Mollicutes

Mycoplasma is the smallest self-replicating bacteria, figuring as common contaminant of eukaryotic cell cultures. Production inputs and operator’s manipulation seem to be the main sources of such contamination. Many analytical approaches have been applied for mycoplasma detection in cell cultures and also in biological products. However, unless they were validated, only indicator cell culture and bacteriological culture are considered as compendial methods for quality control of biological products. Nano-flow cytometry has been pointed out as an alternative technique for addressing prokaryotic and eukaryotic cell viability being a substantial tool for reference material production.

In this study, a viability-flow-cytometry assay was standardized for M. gallisepticum and then applied to other cell-culture-contaminant mycoplasmas. For this, M. galliseticum’s growth rate was observed and different treatments were evaluated to establish low viability cultures (cell death-induced control). Distinct viability markers and their ideal concentrations (titration) were appraised. Ethanol treatment showed to be the best death-inducing control. CFDA and TOPRO markers revealed to be the best choice for detecting live and dead mycoplasma frequencies, respectively.

The standardized methodology was applied to Mycoplasma arginini, M. hyorhinis, M. orale, Spiroplasma citri and Acholeplasma laidlawii. Significant statistical difference was observed in the percentage of viable cells in comparison to ethanol treatment for A. laidlawii in CFDA and in both markers for M. gallisepticum, M. hyorhinis and S. citri. In summary, we standardized a flow cytometry assay for assessing M. gallisepticum – and potentially other species – viability and ultimately applied for reference material production improving the quality control of biological products.

Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis

Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods. Karyotype reports of chondrichthyans are scarce because of the difficulty in cell culture.

Here, we focused on carpet shark species and the culture conditions for fibroblasts and lymphocytes. The utility of the cultured cells enabled the high-fidelity characterization of their karyotypes, namely 2n = 102 for the whale shark (Rhincodon typus) and zebra shark (Stegostoma fasciatum), and 2n = 106 for the brownbanded bamboo shark (Chiloscyllium punctatum) and whitespotted bamboo shark (C. plagiosum).

We identified heteromorphic XX/XY sex chromosomes for the two latter species and demonstrated the first-ever fluorescence in situ hybridization of shark chromosomes prepared from cultured cells. Our protocols are applicable to diverse chondrichthyan species and will deepen the understanding of early vertebrate evolution at the molecular level.