Leukemic Stem Cell Culture in Cytokine-Free Medium

eaaci2015

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 A2a receptor (A2aR) 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 A2aR selective antagonists, CPT and ZM 241385. Furthermore, knockdown of A1R and A2aR 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 A2aR-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.
Paraffin Wax Dispenser
Paraffin wax, granular (56 - 60)
Paraffin wax, granular (56 - 60)
MagSi-WAX
MagSi-WAX
MagSi-WAX
Wax Paper For Se235 Pkg100
neo WAX 30mg Plate SPE Columns
neo WAX 100mg/3mL SPE Columns
neo WAX 200mg/6mL SPE Columns

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

eaaci2015

DAPI (Molecular Biology Grade)

CE118 25 mg
EUR 159.6

DAPI (Molecular Biology Grade)

CE119 100 mg
EUR 382.8

Dimethylsulfoxide (Molecular Biology Grade)

CE120 100 ml
EUR 66

Dimethylsulfoxide (Molecular Biology Grade)

CE121 500 ml
EUR 110.4

DTT (Molecular Biology Grade)

CE131 5 g
EUR 93.6

DTT (Molecular Biology Grade)

CE132 10 g
EUR 133.2

DTT (Molecular Biology Grade)

CE133 25 g
EUR 243.6

Glycine (Molecular Biology Grade)

CE158 1 kg
EUR 84

Glycine (Molecular Biology Grade)

CE159 5 kg
EUR 228

HEPES (Molecular Biology Grade)

CE171 100 g
EUR 98.4

HEPES (Molecular Biology Grade)

CE172 500 g
EUR 268.8

HEPES (Molecular Biology Grade)

CE173 1 kg
EUR 424.8

Lysozyme (Molecular Biology Grade)

CE188 1 g
EUR 70.8

Lysozyme (Molecular Biology Grade)

CE189 10 g
EUR 247.2

NAD (Molecular Biology Grade)

CE196 1 g
EUR 72

NAD (Molecular Biology Grade)

CE197 5 g
EUR 165.6

NBT (Molecular Biology Grade)

CE209 1 g
EUR 123.6

NBT (Molecular Biology Grade)

CE210 5 g
EUR 360

Tris (Molecular Biology Grade)

CE237 500 g
EUR 106.8

Tris (Molecular Biology Grade)

CE238 1 kg
EUR 153.6

Tris (Molecular Biology Grade)

CE239 5 kg
EUR 535.2

Tween20 (Molecular Biology Grade)

CE242 1 l
EUR 106.8

Water (Molecular Biology Grade)

CE243 500 ml
EUR 62.4

Water (Molecular Biology Grade)

CE244 1 l
EUR 67.2

100mL Molecular Biology Grade

46-000-CI PK6
EUR 70.68

500mL Molecular Biology Grade

46-000-CV PK6
EUR 124.26

Ammonium sulfate (Molecular Biology Grade)

CE105 250 g
EUR 55.2

Ammonium sulfate (Molecular Biology Grade)

CE106 1 kg
EUR 72

Ammonium sulfate (Molecular Biology Grade)

CE107 5 kg
EUR 153.6

Bis-Acrylamid (Molecular Biology Grade)

CE110 50 g
EUR 94.8

Bis-Acrylamid (Molecular Biology Grade)

CE111 250 g
EUR 259.2

Formamide deionized (Molecular Biology Grade)

CE145 500 ml
EUR 87.6

Formamide deionized (Molecular Biology Grade)

CE146 1 l
EUR 120

Glycerol 87 % (Molecular Biology Grade)

CE154 1 l
EUR 93.6

Glycerol waterfree (Molecular Biology Grade)

CE155 500 ml
EUR 78

Glycerol waterfree (Molecular Biology Grade)

CE156 1 l
EUR 102

Glycerol waterfree (Molecular Biology Grade)

CE157 2.5 l
EUR 170.4

Guanidine - Hydrochloride (Molecular Biology Grade)

CE160 100 g
EUR 93.6

Guanidine - Hydrochloride (Molecular Biology Grade)

CE161 250 g
EUR 153.6

Guanidine - Hydrochloride (Molecular Biology Grade)

CE162 500 g
EUR 232.8

Guanidine - Hydrochloride (Molecular Biology Grade)

CE163 1 kg
EUR 352.8

Guanidine Thiocyanate (Molecular Biology Grade)

CE164 100 g
EUR 86.4

Guanidine Thiocyanate (Molecular Biology Grade)

CE165 500 g
EUR 192

Guanidine Thiocyanate (Molecular Biology Grade)

CE166 1 kg
EUR 307.2

Urea Crystalline (Molecular Biology Grade)

CE167 1 kg
EUR 72

Urea Crystalline (Molecular Biology Grade)

CE168 5 kg
EUR 181.2

MOPS buffer (Molecular Biology Grade)

CE194 100 g
EUR 102

MOPS buffer (Molecular Biology Grade)

CE195 250 g
EUR 169.2

Sodium chloride (Molecular Biology Grade)

CE205 500 g
EUR 62.4

Sodium chloride (Molecular Biology Grade)

CE206 1 kg
EUR 70.8

Sodium chloride (Molecular Biology Grade)

CE207 5 kg
EUR 123.6

D(+)-Sucrose (Molecular Biology Grade)

CE224 500 g
EUR 67.2

D(+)-Sucrose (Molecular Biology Grade)

CE225 1 kg
EUR 84

D(+)-Sucrose (Molecular Biology Grade)

CE226 5 kg
EUR 207.6

Tris - Hydrochloride (Molecular Biology Grade)

CE234 250 g
EUR 99.6

Tris - Hydrochloride (Molecular Biology Grade)

CE235 500 g
EUR 144

Tris - Hydrochloride (Molecular Biology Grade)

CE236 1 kg
EUR 223.2

TritonX-100 (Molecular Biology Grade)

CE240 500 ml
EUR 67.2

TritonX-100 (Molecular Biology Grade)

CE241 1 l
EUR 79.2

Water, Ultrapure Molecular Biology Grade

41024-4L 4L
EUR 145.2
Description: Minimum order quantity: 1 unit of 4L

Urea, suitable for molecular biology

GE1210-1KG 1 kg
EUR 106.8

Urea, suitable for molecular biology

GE1210-500G 500 g
EUR 76.8

Tween 20, Molecular Biology Grade

T9100-010 100ml
EUR 86.4

Tween 20, Molecular Biology Grade

T9100-050 500ml
EUR 133.2

Tween 20, Molecular Biology Grade

T9100-100 1L
EUR 160.8

1L Molecular Biology Grade Water

46-000-CM PK6
EUR 177.84

EDTA - Dinatriumsalz - Dihydrat (Molecular Biology Grade)

CE135 250 g
EUR 72

EDTA - Dinatriumsalz - Dihydrat (Molecular Biology Grade)

CE136 500 g
EUR 86.4

EDTA - Dinatriumsalz - Dihydrat (Molecular Biology Grade)

CE137 1 kg
EUR 124.8

EDTA - Dinatriumsalz - Dihydrat (Molecular Biology Grade)

CE138 5 kg
EUR 418.8

D(+)-Glucose waterfree (Molecular Biology Grade)

CE148 500 g
EUR 67.2

D(+)-Glucose waterfree (Molecular Biology Grade)

CE149 1 kg
EUR 75.6

D(+)-Glucose waterfree (Molecular Biology Grade)

CE150 5 kg
EUR 180

Yeast extract powder (Molecular Biology Grade)

CE169 500 g
EUR 133.2

Hyaluronidase Grade I (Molecular Biology Grade)

CE174 1 g
EUR 232.8

Hyaluronidase Grade I (Molecular Biology Grade)

CE175 5 g
EUR 920.4

Magnesium acetate - Tetrahydrate (Molecular Biology Grade)

CE190 500 g
EUR 98.4

NADH - Disodium salt (Molecular Biology Grade)

CE198 1 g
EUR 91.2

NADH - Disodium salt (Molecular Biology Grade)

CE199 5 g
EUR 244.8

NADP - sodium salt (Molecular Biology Grade)

CE200 250 mg
EUR 92.4

NADP - sodium salt (Molecular Biology Grade)

CE201 1 g
EUR 190.8

NADPH - Tetrasodium salt (Molecular Biology Grade)

CE202 25 mg
EUR 70.8

NADPH - Tetrasodium salt (Molecular Biology Grade)

CE203 100 mg
EUR 126

NADPH - Tetrasodium salt (Molecular Biology Grade)

CE204 500 mg
EUR 374.4

SSC Buffer (20X) (Molecular Biology Grade)

CE229 1 l
EUR 86.4

XTT sodium salt (Molecular Biology Grade)

CE250 100 mg
EUR 208.8

XTT sodium salt (Molecular Biology Grade)

CE251 500 mg
EUR 612

DiscoveryProbe? Cancer Biology-related Compounds Panel

L1003-5 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 1 kg
EUR 90

Albumin fraction V (pH7,0) (Molecular Biology Grade)

CE100 50 g
EUR 128.4

Albumin fraction V (pH7,0) (Molecular Biology Grade)

CE101 100 g
EUR 193.2

Albumin fraction V (pH7,0) (Molecular Biology Grade)

CE102 250 g
EUR 387.6

Albumin fraction V (pH7,0) (Molecular Biology Grade)

CE103 500 g
EUR 656.4

Albumin fraction V (pH7,0) (Molecular Biology Grade)

CE104 1 kg
EUR 1162.8

Dendritic Cells/B Cells Antibody

abx412775-02mg 0.2 mg
EUR 678

anti-CD45 RA B-cells, T-cells, NK-cells

516-A-01mg 0,1 mg
EUR 321
Description: anti-CD45 RA B-cells, T-cells, NK-cells

anti-CD45 RA B-cells, T-cells, NK-cells

516-A-1000ug 1000 ug
EUR 1539
Description: anti-CD45 RA B-cells, T-cells, NK-cells

 

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 CellCulture-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.
Paraffin Wax Dispenser
Paraffin wax, granular (56 - 60)
Paraffin wax, granular (56 - 60)
MagSi-WAX
MagSi-WAX
MagSi-WAX
Wax Paper For Se235 Pkg100
neo WAX 30mg Plate SPE Columns
neo WAX 100mg/3mL SPE Columns
neo WAX 200mg/6mL SPE Columns