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ClonaCell™tcs介质

半固体甲基纤维素培养基，用于选择和克隆悬浮适应细胞和贴壁细胞（含血清）
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产品号 #（选择产品）

产品号 #03814_C

半固体甲基纤维素培养基，用于选择和克隆悬浮适应细胞和贴壁细胞（含血清）

产品优势

  • 支持多种细胞系的高克隆效率和强大的集落形成，如：B16F-10， BaF/3, bkh -1, CHO-DG44, CHO-K1, CHO-S, FD-5, HEK-293, Jurkat Daudi, K562, Molt-4, UT-7
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概述

ClonaCell™-TCS Medium是一种含血清甲基纤维素的半固体培养基，可用于选择和克隆多种悬浮适应细胞系，包括CHO-S和杂杂瘤。它也可用于在血清存在下粘附生长的细胞系的半固体克隆，包括CHO-K1、BHK-1和HEK-293。这种培养基含有预先选择的胎牛血清（FBS）和牛血清白蛋白（BSA），支持多种细胞类型的强劲生长。ClonaCell™-TCS Medium不含选择剂。

半固态克隆的好处：
•单个细胞悬浮在粘性介质中，形成物理分离、离散的菌落，易于分离。
•与选择和克隆相比，通过限制稀释，单克隆细胞系在更短的时间内使用更少的资源进行分离。
•不同的克隆具有广泛的生长速率和生产力，在粘性介质中形成离散的菌落。因此，在半固体培养基中同时选择和克隆比在散装液体培养基中选择更容易单独分离稀有和高产无性系。

Contains
• IMDM (Iscove's Modified Dulbecco's Medium)
• Methylcellulose
• Pre-selected serum
• Bovine serum albumin
• 2-Mercaptoethanol
• Phenol red
• L-Glutamine
• Other ingredients
 
Subtype
Semi-Solid Media, Specialized Media
 
Cell Type
CHO Cells, HEK-293, Hybridomas, Other
 
Species
Mouse, Other
 
Application
Cell Culture, Semi-Solid Cloning
 
Brand
ClonaCell
 
Area of Interest
Antibody Development, Cell Line Development, Hybridoma Generation
 

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Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Document Type
Product Information Sheet
Product Name
ClonaCell™-TCS Medium
Catalog #
03814
Lot #
All
Language
English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Research Area
Workflow Stages

Resources and Publications

Educational Materials (6)

ClonaCell-CHO Semi-Solid Cloning Testing Guidelines
Brochure
ClonaCell-CHO Semi-Solid Cloning Testing Guidelines
ClonaCell™ Media for Hybridoma and Cell Line Development
Brochure
ClonaCell™ Media for Hybridoma and Cell Line Development
Protocol for Cloning CHO Cell Lines Using ClonaCell™-CHO Semi-Solid Medium
Technical Bulletin
Protocol for Cloning CHO Cell Lines Using ClonaCell™-CHO Semi-Solid Medium
Semi-Solid Cloning of CHO Cells Using ClonaCell™-CHO Medium
10:50
Video
Semi-Solid Cloning of CHO Cells Using ClonaCell™-CHO Medium
Why Use Semi-Solid Medium for Cloning Hybridomas and CHO Cells
3:39
Video
Why Use Semi-Solid Medium for Cloning Hybridomas and CHO Cells
How to Perform Semi-Solid Cloning of CHO Cell Lines in 96-Well Plates Using ClonaCell™-CHO Medium
12:47
Video
How to Perform Semi-Solid Cloning of CHO Cell Lines in 96-Well Plates Using ClonaCell™-CHO Medium

Frequently Asked Questions

Is the serum in ClonaCell™-TCS medium heat inactivated?

Yes, all serum used in ClonaCell™ is heat inactivated.

Is there any IgG in ClonaCell™ TCS?

While we don't add IgG to the ClonaCell™ media, we do add serum, which contains an undefined amount of IgG. We selectively use serum lots with low IgG levels in the production of ClonaCell™ media, however, levels vary from lot to lot. IgG levels in a specific lot of ClonaCell™ TCS medium are available in the lot-specific Certificate of Analysis.

Can ClonaCell™-TCS be used with any cell line?

A list of recommended cell lines can be found in the manual. Other cell lines may be compatible with ClonaCell™-TCS. It will be necessary, however, to determine the plating cell density and growth efficiency of the desired cells in ClonaCell™-TCS.

Why do I get more cells when I select my fusion in liquid medium rather than in methylcellulose-based semi-solid medium?

Cells grown in liquid medium may appear to grow more rapidly than in methylcellulose-based medium. This is often due to the presence of a few rapidly growing clones that multiply quickly and become abundant in liquid culture, overgrowing clones that grow more slowly. In methylcellulose cultures, the rapidly growing cells remain in close proximity to each other, resulting in large colonies, each derived from a single fusion or transfection product. The large clones don't overgrow smaller, slower growing colonies, which can be separately isolated.

How do I thaw ClonaCell™ methylcellulose-based semi-solid medium?

We recommend thawing the medium overnight in a refrigerator at 4°C and mixing well.

How do I measure and dispense methylcellulose semi-solid medium?

We recommend using a 12 mL syringe with a 16 gauge needle attached (blunt end needles are recommended for safety purposes). Do not dispense the semi-solid media/cell mixture using serological pipettes as the media will stick to the pipette walls, resulting in inaccurate dispensed volumes and loss of cells.

My ClonaCell™ methylcellulose semi-solid medium appears runny. Why does this happen?

"Runny" methylcellulose could be a result of improper handling. Diluting the methylcellulose with too much liquid medium, or insufficient mixing before use, will result in methylcellulose with altered viscosity. Excessive condensation on the inside of the cell culture dish lid can result in water dripping onto the cultures, lowering viscosity. Additionally, bumping, shaking or other sudden movement of the culture may also disrupt the colonies. Note: methylcellulose is less viscous at room temperature than at 37°C.

What is the optimal number of colonies per plate?

We recommend 50-150 colonies per plate. As it is difficult to anticipate the numbers of colonies after a fusion or transfection, we recommend plating at three different densities to increase the likelihood of achieving a plating density of approximately 100 colonies per plate. This density allows sufficient space between the colonies to allow for easy colony picking.

There are still bubbles in the media after I plate my cells. Do I need to disrupt the bubbles?

We recommend that you avoid creating large bubbles during plating, but there is no need to manually pop or disperse the small bubbles after plating. They will disperse over the incubation period of 10-14 days.

Do I ever need to re-clone cultures grown with ClonaCell™ semi-solid medium?

Re-cloning is a good practice to observe and is recommended if the number of colonies in the original dishes was very high.

Once I pick the colonies and grow the cells in plates, will the residual methylcellulose interfere with characterization? For example, will I have problems doing an ELISA?

There will likely be some residual methylcellulose contamination when colonies are picked and transferred to the 96-well plate with the liquid growth medium. The concentration of methylcellulose, however, should be low enough that it should not interfere with most assays.

How important is the incubator humidity when culturing in methylcellulose-based medium?

Very important. In situations where the humidity is not high enough, we recommend that the 100 mm Petri dishes should be placed with an open dish containing sterile water inside a larger plastic container with a lid. Without very high humidity, the media will dry out over the culture period and this will impede the growth of the colonies.

Do I have to use 100 mm petri dishes or can I use other cultureware?

We recommend 100 mm Petri dishes as these have been used to develop and test ClonaCell™ semi-solid media. We have found that the surface area of these dishes allows for easy colony picking. Other sizes of dish (e.g. 6-well plates) can be used. It is important to use non-coated dishes to prevent cells from sticking to the bottom of the plate and obscuring the colonies. The volume of media plated should be adjusted to reflect the surface area of the dish being used.

Publications (12)

The T-cell leukemia-associated ribosomal RPL10 R98S mutation enhances JAK-STAT signaling. T. Girardi et al. Leukemia 2018 MAR

Abstract

Several somatic ribosome defects have recently been discovered in cancer, yet their oncogenic mechanisms remain poorly understood. Here we investigated the pathogenic role of the recurrent R98S mutation in ribosomal protein L10 (RPL10 R98S) found in T-cell acute lymphoblastic leukemia (T-ALL). The JAK-STAT signaling pathway is a critical controller of cellular proliferation and survival. A proteome screen revealed overexpression of several Jak-Stat signaling proteins in engineered RPL10 R98S mouse lymphoid cells, which we confirmed in hematopoietic cells from transgenic Rpl10 R98S mice and T-ALL xenograft samples. RPL10 R98S expressing cells displayed JAK-STAT pathway hyper-activation upon cytokine stimulation, as well as increased sensitivity to clinically used JAK-STAT inhibitors like pimozide. A mutually exclusive mutation pattern between RPL10 R98S and JAK-STAT mutations in T-ALL patients further suggests that RPL10 R98S functionally mimics JAK-STAT activation. Mechanistically, besides transcriptional changes, RPL10 R98S caused reduction of apparent programmed ribosomal frameshifting at several ribosomal frameshift signals in mouse and human Jak-Stat genes, as well as decreased Jak1 degradation. Of further medical interest, RPL10 R98S cells showed reduced proteasome activity and enhanced sensitivity to clinical proteasome inhibitors. Collectively, we describe modulation of the JAK-STAT cascade as a novel cancer-promoting activity of a ribosomal mutation, and expand the relevance of this cascade in leukemia.
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DNMT3A mutations promote anthracycline resistance in acute myeloid leukemia via impaired nucleosome remodeling. Guryanova OA et al. Nature Medicine 2016 NOV

Abstract

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3A(R882)), have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3A(R882) AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy, suggesting that DNMT3A(R882) cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3(ITD)) and the nucleophosmin gene (Npm1(c)) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3A(R882) mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3A(R882) cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3A(R882) mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.
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Generation of the Fip1l1–Pdgfra fusion gene using CRISPR/Cas genome editing Vanden Bempt M et al. Leukemia 2016 MAR

Abstract

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Species Mouse, Other
Contains • IMDM (Iscove's Modified Dulbecco's Medium) • Methylcellulose • Pre-selected serum • Bovine serum albumin • 2-Mercaptoethanol • Phenol red • L-Glutamine • Other ingredients
PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT STEMCELL, REFER TO WWW.STEMCELL.COM/COMPLIANCE.
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