Description

CL990-cin is a selection antibiotic that acts on both eukaryotic and prokaryotic cells. Resistance
to CL990-cin is conferred by the Sh ble gene from Streptoalloteichus hindustanus1-3. CL990-cin
is the commercial name for a special formulation containing Phleomycin, a copper-chelated glycopeptide antibiotic isolated from a mutant strain of Streptomyces verticillus. This antibiotic of the bleomycin family exhibits activity against bacteria, eukaryotic microorganisms, plant and animal cells. Although bleomycin antibiotics perturb plasma membranes, their activity is generally believed to be related to their ability to bind and intercalate DNA thus destroying the integrity of the double helix

Chemical Properties

CL990-cin is a mixture of structurally related antibiotics which differ by their terminal amine residues. The antibiotics are in a copper chelated form giving the solution a blue color. CL990-cin is a labile compound which undergoes irreversible denaturation at high and low pH or in presence of a weak oxidant. CL990-cin is freely soluble in water (>500 mg/ml) forming a blue solution.

Preparation

Resuspend CL990-cin in HEPES buffer (5 g/l, pH 7.2+/- 0.1) at a concentration of 100 mg/ml.

Sterile filter the solution using a 0.22μm sterile filter.

Store at 4°C for 12 months or -20 °C for 18 months.

General Guidelines

Successful transfection is influenced by many factors. The health and viability of the cell line, the quality of the nucleic acid used, the transfection reagent, the duration of transfection, and the presence or absence of serum can all play a part.

Condition of Selection

Most cells growing aerobically are killed by CL990-cin in the concentration range of 0.5 to 1000 μg/ml. However, the sensitivity of cells is pH dependent, i.e. the higher the pH of culture medium,the greater the sensitivity. Thus, the concentration of CL990-cin required for complete growth inhibition of given cells can be reduced by increasing the pH of the medium. In addition, the activity of CL990-cin is reduced by a factor of two to three in hypertonic media, such as those used for protoplast regeneration. Thus, using low salt media when possible decreases the amount of CL990-cin needed.

- Escherichia coli

The Sh ble gene and the hybrid genes in vectors provided by InvivoGen are driven by synthetic
E. coli promoters (i.e. EM7). The cells of the common E. coli recipient strains (i.e. HB101, DH5a,MC1061) transformed by these vectors are resistant to CL990-cin

Note: Do not use an E. coli recipient strain that contains the Tn5 transposable element (i.e. MC1066). Tn5 encodes a bleomycinresistance gene that will confer resistance to CL990-cin.

CL990-cin -resistant transformants are selected in Low Salt LB agar medium (yeast extract 5g/l,
Tryptone 10g/l, NaCl 5g/l, Agar 15 g/l, pH 7.5) supplemented with 25 μg/ml of CL990-cin. Plates
containing CL990-cin are stable for 1 month when stored at 4°C.

- Mammalian cells

The working concentration of CL990-cin for mammalian cell lines varies from 50 to 400 μg/ml, in a few cases can be as low as 20 μg/ml or as high as 1000 μg/ml. In a starting experiment we recommend to determine the optimal concentration of CL990-cin required to kill your host cell line. The killing and the detachment of dead cells from the plate, especially at high cell density, can require a longer time compared to G418. Foci of CL990-cin -resistant stable transfectants are usually individualized after 5 days to 3 weeks incubation, depending on the cell line. Suggested concentrations of CL990-cin for selection in mammalian cells are listed on the next page

Working Concentration

CL990-cin is normally used at a concentration of 100 μg/ml, a 1000-fold dilution from the stock
solution. However, the optimal concentration needs to be determined for your cells. Suggested
concentrations of CL990-cin for selection in some examples of mammalian cells are listed below.

Cell line	Medium	CL990-cin conc	References
B16 (Mouse melanocytes)	RPMI	20-250 µg/ml	4-6
CHO (Chinese hamster ovarian cells)	DMEM	100-500 µg/ml	4, 7, 8
COS (Monkey kidney cells)	DMEM	100-400 µg/ml	9, 10
HEK293 (Human embryonic kidney cells)	DMEM	100-400 µg/ml	11, 12
HeLa (Human uterine cells)	DMEM	50-100 µg/ml	13, 14
J558L (Mouse melanocytes)	RPMI	400 µg/ml	15
MCF-7 (Human breast adenocarcinoma cells)	DMEM	100-400 µg/ml	16, 17
MEFs (Mouse embryonic fibroblasts)	DMEM	200-400 µg/ml	18, 19
THP-1 (Human monocytes)	RMPI	200 µg/ml	20

Storage/Stability

CL990-cin is shipped at room temperature. Upon receipt it should be stored at 4°C. The expiry
date is specified on the product label.

Store CL990-cin solutions at 4°C or -20°C. Do not use CL990-cin solutions after the expiry
date on the product label. Avoid repeated freeze-thaw cycles.

CL990-cin is sensitive to high concentrations of acids and bases but a short-term exposure to
dilute acids can be tolerated.

Quality Control

Each lot is tested to ensure the absence of lot-to-lot variation.

Endotoxin level: < 1 EU/mg

Physicochemical characterization (including HPLC, pH, appearance)

Cell culture tested: potency validated in CL990-cin -sensitive and CL990-cin -resistant
mammalian cell lines

Non-cytotoxicity of trace contaminants: absence of long-term effects confirmed in CL990-cin -
resistant cells

Precautions and Disclaimer

CL990-cin is a harmful compound. Refer to safety data sheet for handling instructions

Documents

INFO

SDS

Disclaimer: For laboratory research use only.