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 A
1R 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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Paraffin Wax Dispenser |
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Paraffin wax, granular (56 - 60) |
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Paraffin wax, granular (56 - 60) |
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Paraffin wax, granular (56 - 60) |
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Paraffin wax, granular (56 - 60) |
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8KG Histoplast PE Paraffin |
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Paraffin oil, BP, Ph. Eur. grade |
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Paraffin oil, BP, Ph. Eur. grade |
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Paraffin oil, BP, Ph. Eur. grade |
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Paraffin oil, BP, Ph. Eur. grade |
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
) DTT (Molecular Biology Grade) |
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CE133 |
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25 g |
EUR 243.6 |
) Tris (Molecular Biology Grade) |
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CE237 |
GeneOn |
500 g |
EUR 106.8 |
Tris (Molecular Biology Grade) |
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CE238 |
GeneOn |
1 kg |
EUR 153.6 |
Tris (Molecular Biology Grade) |
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CE239 |
GeneOn |
5 kg |
EUR 535.2 |
) BCIP (Molecular Biology Grade) |
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CE108 |
GeneOn |
250 mg |
EUR 75.6 |
BCIP (Molecular Biology Grade) |
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CE109 |
GeneOn |
1 g |
EUR 108 |
) DAPI (Molecular Biology Grade) |
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CE117 |
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5 mg |
EUR 72 |
DAPI (Molecular Biology Grade) |
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CE118 |
GeneOn |
25 mg |
EUR 159.6 |
DAPI (Molecular Biology Grade) |
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CE119 |
GeneOn |
100 mg |
EUR 382.8 |
 DMSO, Molecular Biology Grade |
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40470006-1 |
Bio-WORLD |
100 mL |
EUR 88.18 |
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DMSO, Molecular Biology Grade |
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40470006-2 |
Bio-WORLD |
250 mL |
EUR 150.19 |
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DMSO, Molecular Biology Grade |
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40470006-3 |
Bio-WORLD |
500 mL |
EUR 279.26 |
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 EGTA, Molecular Biology Grade |
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40500028-2 |
Bio-WORLD |
50 g |
EUR 106.43 |
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EGTA, Molecular Biology Grade |
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40500028-3 |
Bio-WORLD |
100 g |
EUR 177.58 |
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EGTA, Molecular Biology Grade |
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40500028-4 |
Bio-WORLD |
500 g |
EUR 603.19 |
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EGTA, Molecular Biology Grade |
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40500028-5 |
Bio-WORLD |
1 kg |
EUR 912.98 |
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EGTA, Molecular Biology Grade |
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40500028-6 |
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2 kg |
EUR 1687.94 |
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) HEPES (Molecular Biology Grade) |
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CE171 |
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HEPES (Molecular Biology Grade) |
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CE172 |
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HEPES (Molecular Biology Grade) |
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) Water (Molecular Biology Grade) |
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CE243 |
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Water (Molecular Biology Grade) |
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CE244 |
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CE114 |
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CHAPS (Molecular Biology Grade) |
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CE116 |
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25 g |
EUR 492 |
) Tween20 (Molecular Biology Grade) |
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CE242 |
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1 l |
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) Glycine (Molecular Biology Grade) |
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CE158 |
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EUR 84 |
Glycine (Molecular Biology Grade) |
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CE159 |
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EUR 228 |
 Agarose, Molecular Biology Grade |
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40100164-1 |
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25 g |
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Agarose, Molecular Biology Grade |
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40100164-2 |
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50 g |
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Agarose, Molecular Biology Grade |
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40100164-3 |
Bio-WORLD |
100 g |
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Agarose, Molecular Biology Grade |
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40100164-4 |
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500 g |
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Agarose, Molecular Biology Grade |
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40100164-5 |
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1 kg |
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) Lysozyme (Molecular Biology Grade) |
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CE188 |
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1 g |
EUR 70.8 |
Lysozyme (Molecular Biology Grade) |
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CE189 |
GeneOn |
10 g |
EUR 247.2 |
 Tween 20, Molecular Biology Grade |
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T9100-010 |
GenDepot |
100ml |
EUR 86.4 |
Tween 20, Molecular Biology Grade |
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T9100-050 |
GenDepot |
500ml |
EUR 133.2 |
Tween 20, Molecular Biology Grade |
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T9100-100 |
GenDepot |
1L |
EUR 160.8 |
-Sucrose (Molecular Biology Grade)) D(+)-Sucrose (Molecular Biology Grade) |
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CE224 |
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500 g |
EUR 67.2 |
D(+)-Sucrose (Molecular Biology Grade) |
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CE225 |
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EUR 84 |
D(+)-Sucrose (Molecular Biology Grade) |
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CE226 |
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 Boric Acid, Molecular Biology Grade |
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40200060-1 |
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500 g |
EUR 46.16 |
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Boric Acid, Molecular Biology Grade |
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40200060-2 |
Bio-WORLD |
1 kg |
EUR 76.66 |
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Boric Acid, Molecular Biology Grade |
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40200060-3 |
Bio-WORLD |
2.5 kg |
EUR 145.5 |
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) MOPS buffer (Molecular Biology Grade) |
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CE194 |
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MOPS buffer (Molecular Biology Grade) |
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CE195 |
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) Glycerol 87 % (Molecular Biology Grade) |
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CE154 |
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CE120 |
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100 ml |
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Dimethylsulfoxide (Molecular Biology Grade) |
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CE121 |
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500 ml |
EUR 110.4 |
) TritonX-100 (Molecular Biology Grade) |
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CE240 |
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500 ml |
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TritonX-100 (Molecular Biology Grade) |
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CE241 |
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1 l |
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 Water, Ultrapure Molecular Biology Grade |
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41024-4L |
Biotium |
4L |
EUR 145.2 |
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Description: Minimum order quantity: 1 unit of 4L |
) Sodium chloride (Molecular Biology Grade) |
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CE205 |
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500 g |
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Sodium chloride (Molecular Biology Grade) |
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CE206 |
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CE207 |
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CE111 |
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40300060-1 |
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50 g |
EUR 45.77 |
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) Urea Crystalline (Molecular Biology Grade) |
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CE167 |
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Urea Crystalline (Molecular Biology Grade) |
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CE168 |
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CE105 |
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EUR 55.2 |
Ammonium sulfate (Molecular Biology Grade) |
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CE106 |
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Ammonium sulfate (Molecular Biology Grade) |
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CE107 |
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 Lithium Chloride, Molecular Biology Grade |
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41200036-1 |
Bio-WORLD |
100 g |
EUR 41.7 |
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Lithium Chloride, Molecular Biology Grade |
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41200036-2 |
Bio-WORLD |
500 g |
EUR 108.38 |
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 (Molecular Biology Grade)) SSC Buffer (20X) (Molecular Biology Grade) |
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CE229 |
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1 l |
EUR 86.4 |
) Tris - Hydrochloride (Molecular Biology Grade) |
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CE234 |
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250 g |
EUR 99.6 |
Tris - Hydrochloride (Molecular Biology Grade) |
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CE235 |
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500 g |
EUR 144 |
Tris - Hydrochloride (Molecular Biology Grade) |
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CE236 |
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1 kg |
EUR 223.2 |
) Glycerol waterfree (Molecular Biology Grade) |
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CE155 |
GeneOn |
500 ml |
EUR 78 |
Glycerol waterfree (Molecular Biology Grade) |
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CE156 |
GeneOn |
1 l |
EUR 102 |
Glycerol waterfree (Molecular Biology Grade) |
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CE157 |
GeneOn |
2.5 l |
EUR 170.4 |
) XTT sodium salt (Molecular Biology Grade) |
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CE250 |
GeneOn |
100 mg |
EUR 208.8 |
XTT sodium salt (Molecular Biology Grade) |
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CE251 |
GeneOn |
500 mg |
EUR 612 |
) Formamide deionized (Molecular Biology Grade) |
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CE145 |
GeneOn |
500 ml |
EUR 87.6 |
Formamide deionized (Molecular Biology Grade) |
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CE146 |
GeneOn |
1 l |
EUR 120 |
) NADP - sodium salt (Molecular Biology Grade) |
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CE200 |
GeneOn |
250 mg |
EUR 92.4 |
NADP - sodium salt (Molecular Biology Grade) |
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CE201 |
GeneOn |
1 g |
EUR 190.8 |
) Guanidine Thiocyanate (Molecular Biology Grade) |
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CE164 |
GeneOn |
100 g |
EUR 86.4 |
Guanidine Thiocyanate (Molecular Biology Grade) |
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CE165 |
GeneOn |
500 g |
EUR 192 |
Guanidine Thiocyanate (Molecular Biology Grade) |
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CE166 |
GeneOn |
1 kg |
EUR 307.2 |
) NADH - Disodium salt (Molecular Biology Grade) |
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CE198 |
GeneOn |
1 g |
EUR 91.2 |
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.
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Paraffin Wax Dispenser |
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Paraffin wax, granular (56 - 60) |
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Paraffin wax, granular (56 - 60) |
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Paraffin wax, granular (56 - 60) |
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Paraffin wax, granular (56 - 60) |
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8KG Histoplast PE Paraffin |
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Paraffin oil, BP, Ph. Eur. grade |
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Paraffin oil, BP, Ph. Eur. grade |
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Paraffin oil, BP, Ph. Eur. grade |
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Paraffin oil, BP, Ph. Eur. grade |
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