Gibco™ Leibovitz's L-15 Medium, GlutaMAX™ Supplement

Catalog No.	Quantity
31-415-029	500 mL

Catalog No. 31-415-029 Supplier Gibco™ Supplier No. 31415029

Description

Leibovitz's L-15 Medium supports the growth of HEP-2 monkey kidney cells and primary explants of embryonic and adult human tissue. L-15 is buffered by phosphates and free base amino acids instead of sodium bicarbonate.

Leibovitz's L-15 Medium supports the growth of HEP-2 monkey kidney cells and primary explants of embryonic and adult human tissue. L-15 is buffered by phosphates and free base amino acids instead of sodium bicarbonate. This medium is designed for supporting cell growth in environments without CO₂ equilibration.

This L-15 is modified as follows:

With: Galactose, Phenol Red, GlutaMAX™, Sodium Pyruvate

Without: Glucose, HEPES, Sodium Bicarbonate

The complete formulation is available.

Gibco™ L-15 with GlutaMAX™ supplement minimizes toxic ammonia build-up and improves cell viability and growth in an easy-to-use format.

L-15 contains no proteins, lipids, or growth factors. Therefore, L-15 requires supplementation, commonly with 10% Fetal Bovine Serum (FBS).

Specifications

• Classification: Animal Origin-free
• Concentration: 1 X
• Form: Liquid
• Product Type: Leibovitz's L-15 Medium
• Sterility: Sterile-filtered
• With Additives: GlutaMAX, Phenol Red, Sodium Pyruvate, Galactose
• Without Additives: No HEPES, No Sodium Bicarbonate, No Glucose
• Manufacturing Quality: cGMP-compliant under the ISO 13485 standard
• Product Line: Gibco, GlutaMAX
• Quantity: 500 mL
• Shipping Condition: Room Temperature
• Osmolality: 300 - 340 mOsm/kg
• pH: 7.4 to 7.9
• Content And Storage: Storage conditions: 2-8° C. Protect from light.
  Shipping conditions: Ambient.
  Shelf life: 12 months from date of manufacture.
• Culture Environment: Non-CO₂
• Culture Type: Mammalian Cell Culture

Frequently Asked Questions (FAQs)

Is there an alternative for L15 Leibovitz Medium (Cat. Not. 11415056)?

The alternative for L15 Leibovitz Medium (Cat. No. 11415056) is Lebovitz's L-15 Medium, GlutaMAX Supplement (Cat. No. 31415029).

What is the osmolality of Leibovitz's L-15 Medium?

We do provide osmolality information on the certificate of analysis. All lots of Leibovitz's L-15 Medium will meet the osmolality specification of 300-340 mOsm/kg.

What are the Leibovitz media you offer?

We offer the following Leibovitz media:

- Leibovitz's L-15 Medium, powder: Cat. Nos. 41300039 (10x 1L) and 41300070 (1x 10L)
- Leibovitz's L-15 Medium, no phenol red: Cat. No 21083027 (1x 500 mL)
- Leibovitz's L-15 Medium: Cat. Nos.11415064 (1x 500 mL) and 11415114 (10x 500 mL) (for sale in N. America, Latin America, and Asia-Pacific regions; Cat. No. 11415049 (for sale in Europe, Africa, and the Middle East
- Leibovitz's L-15 Medium with GlutaMAX Supplement: Cat. No. 31415029 (1x 500 mL)

How long can I keep my media after supplementing with serum?

Generally speaking, media can be used for up to three weeks after supplementation with serum. There are no formal studies to support this, but it is the rule of thumb used by our scientists.

My medium was shipped at room temperature but it is supposed to be stored refrigerated. Is it okay?

We routinely ship media that require long-term storage in the refrigerator at room temperature. We have done studies on representative media formulations to show that media can be at room temperature for up to a week without a problem.

Will depletion, absence, or breakdown of essential growth-promoting components such as glutamine or growth factors reduce the growth rate of my culture?

Yes. If you suspect that this is the case, remove the medium and add fresh medium. Alternatively, you can supplement medium with growth-promoting components. It is also possible to substitute GlutaMax I or II for glutamine in the medium to prevent glutamine exhaustion.

Will the media formulations containing GlutaMAX supplement change with respect to L-glutamine content?

In all media containing GlutaMAX supplement dipeptides as a substitute for L-glutamine, concentration is equimolar with the L-glutamine in the original formulation.

How can I remove mycoplasma contamination from my cell culture medium?

Very often mycoplasma contamination cannot be removed from the culture so it should be discarded. You may have a unique culture that you prefer not to discard and would like to try to clean it. Ciprofloxacin and Plasmocin have reportedly been used for this application. If interested in a protocol or directions for use, check with the antibiotic supplier or published literature. Note that mycoplasma are very difficult to remove from culture and spread easily so the treated cultures should be quarantined until clear of mycoplasma, and your laboratory should be thoroughly cleaned.

I see a decrease in growth of my culture. What should I do?

Try changing the medium or serum. Compare media formulations for differences in glucose, amino acids, and other components. Compare an old lot of serum with a new lot. Increase initial cell inoculums. Lastly, adapt cells sequentially to new medium.

My cells are not adhering to the culture vessel. What should I do?

This can occur if cells are overly trypsinized. Trypsinize for a shorter time or use less trypsin. Mycoplasma contamination could also cause this problem. Segregate your culture and test for mycoplasma infection. Lastly, check for attachment factors in the medium.

I see a precipitate in my medium, and a change in the pH. What should I do?

This is most likely a sign of bacterial or fungal contamination. We would recommend discarding the media, and trying to decontaminate the culture.

I see a precipitate in my medium, but no change in the pH. What should I do?

The precipitate could be due to residual phosphate left over from detergent washing, which may precipitate powered medium components. Rinse glassware in deionized, distilled water several times, then sterilize. If the medium is frozen, try warming media to 37 degrees C and swirl to dissolve. If precipitate remains, discard medium.

I'm seeing a rapid pH shift in my medium. What should I do?

Please see the possible causes for this rapid pH shift, and suggested solutions:

- Incorrect carbon dioxide tension: Increase or decrease percentage of carbon dioxide in the incubator based on concentration of sodium bicarbonate in the medium. For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, use carbon dioxide amounts of 5-10%, respectively. Switch to carbon dioxide-independent medium.
- Overly tight caps on tissue culture flasks: Loosen caps one-quarter turn.
- Insufficient bicarbonate buffering: Add HEPES buffer to a final concentration of 10-25 mM.
- Incorrect salts in medium: Use an Earle's salts-based medium in a CO² environment and a Hanks' salts-based medium in atmospheric conditions.
- Bacterial, yeast, or fungal contamination: Discard culture and medium. Try to decontaminate culture.

My cells are growing very slowly. What could be the cause of this?

Please see some common reasons below, and solutions to the issue:

- Growth medium is not correct: Use pre-warmed growth medium as recommended by the supplier.
- Serum in the growth medium is of poor quality: Use serum from a different lot.
- Cells have been passaged too many times: Use healthy, low-passage number cells.
- Cells were allowed to grow beyond confluency: Passage mammalian cells when they are in the log-phase before they reach confluency.
- Culture is contaminated with mycoplasma: Discard cells, media, and reagents. Obtain new stocks of cells, and use them with fresh media and reagents.

I have no viable cells after thawing my stock. What could be the cause of this?

Please see the possible causes and solutions we recommend below:

- Cells were stored incorrectly: Obtain a new stock and store in liquid nitrogen. Keep cells in liquid nitrogen until thawing.
- Homemade freezer stock is not viable: Freeze cells at a density recommended by the supplier. Use low-passage cells to make your own freezer stocks. Follow procedures for freezing cells exactly as recommended by the supplier. Obtain a new stock.
- Cells were thawed incorrectly: Follow procedures for thawing cells exactly as recommended by the supplier. Make sure that you thaw the frozen cells quickly but dilute them slowly using pre-warmed growth medium before plating.
- Thawing medium is not correct: Use the medium recommended by the supplier. Make sure the medium is pre-warmed.
- Cells are too dilute: Plate thawed cells at the highest density recommended by the supplier to optimize recovery.
- Cells not handled gently: Freezing and thawing procedures are stressful to most cells. Do not vortex, bang the flasks to dislodge the cells (except when culturing insect cells), or centrifuge cells at high speeds.
- Glycerol used in the freezing medium was stored in light (if applicable): If stored in light, glycerol gets converted into acrolein, which is toxic to cells. Obtain a new stock.

What factors can contribute to rapid cell death/culture failure?

There are a number of events that can contribute to this:

1. Incorrect CO² levels-monitor the level of CO² manually with a Fyrite kit, available from Bacharach (http://www.bacharach-inc.com/fyrite_analyzers.htm). Check if the manual readings concur with the readings displayed on the incubator. If the incubator has a trace readout, check the printout for fluctuations in CO² level. Check the settings to insure that CO² levels are set at appropriate levels for your cell line (usually between 5 and 10%). Check line connections frequently for leaks. Avoid frequent opening and closing of incubator doors.
2. Temperature fluctuations in the incubator-Monitor the temperature of incubator with a good thermometer inside the incubator.
3. Fungizone or other preventive antibiotics/antimycotics are present at toxic concentrations-use at recommended levels.
4. Humidity is incorrect-check the water level in the water pan. Humidity is vital to appropriate gas exchange for many types of cells and media (i.e., appropriate CO² levels are largely irrelevant for most cultures if the humidity level is not high enough).
5. Incorrect osmotic pressure in medium-check osmolality of complete medium. Most mammalian cells can tolerate an osmolality of 260 to 350 mOsm/kg. Additions of reagents such as HEPES and drugs may affect osmolality.
6. Contamination by microorganisms-bacterial and fungal contaminations are usually easily visible; symptoms of mycoplasma contamination are more subtle, and careful monitoring of culture morphology and regular testing are necessary to detect this type of contamination.
7. Inappropriate medium is being used-double-check that the medium used is appropriate for your cell type and culture application. For example, ensure that the medium being used for serum-free culture is actually designed for serum-free culture; make sure that appropriate selective drugs are used at appropriate levels; check the expiration dates for the reagents being used; and store medium at appropriate temperatures in the dark.

Based on the sodium bicarbonate levels in the medium, what CO2 percentage is recommended?

If the media formulation contains:
- NaHCO³ (g/L) <1.5, it needs CO² at 4%
- NaHCO³ (g/L) 1.5-2.2, it needs CO² at 5%
- NaHCO³ (g/L) 2.2-3.4, it needs CO² at 7%
- NaHCO³ (g/L) >3.5, it needs CO² at 10%

However, there are some exceptions. Gibco DMEM has always been made according to Dulbecco's original published formulation, with 3.7 g/L sodium bicarbonate. Customers have been using this medium in CO² incubators ranging from 5-10% CO² for decades, usually with no trouble maintaining physiological pH; this also depends on the cell type. Once cells are growing, the pH will drop (due to metabolic accumulation of lactic acid).

How much serum should I add to my media?

The serum concentration will vary with the cell line and basal medium used. Please go here to see our recommended sera supplementations for tested cell lines (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media/advanced-d-mem-and-mem/recommended-sera-supplementations.html).

Do you offer assistance with media formulation development?

Yes. Our Gibco Custom Media Specialists and R&D staff can provide advice related to the addition or elimination of media components. For more comprehensive services, our PD-Direct Bioprocess Services team specializes in media development and optimization.

Who can answer questions related to the Gibco Media Configurator?

We have a dedicated team of Gibco Custom Media Specialists who can partner with you during customization, manufacturing, and delivery. Please email custommedia@lifetech.com or call the local phone number listed on www.thermofisher.com/mediaconfigurator with inquiries about your submission. For questions related to website effectiveness, please contact System Support through the toll-free or local phone number.

What are the benefits of using the Gibco Media Configurator?

Using the Gibco Media Configurator, you can view specific customization options for your cell culture product, online, at any time. Also, this tool provides faster pricing for custom media, generally within 48 business hours.

Can the Gibco Media Configurator be used for my own proprietary formulation?

No, the Gibco Media Configurator cannot be used to request pricing on non-Gibco cell culture products. However, we are very experienced at manufacturing customer-owned media formulations. Please visit www.thermofisher.com/custommedia or contact us at custommedia@lifetech.com for more information.

What aspects of cell culture media can be customized?

The Gibco Media Configurator allows you to add, remove, and adjust the concentration of components. In addition, you can select from a range of packaging, QC tests, and either cGMP or non-cGMP manufacturing.

Can any Gibco formulation be customized with the Gibco Media Configurator?

Almost all Gibco cell culture basal media products can be customized using the Gibco Media Configurator. To check on a particular product, use the Media Configurator page (https://www.thermofisher.com/us/en/home/life-science/bioproduction/custom-cell-culture-media-and-services/gibco-custom-media-configurator.html to search by Gibco catalog number, or browse our offering by description. For the customization of non-media Gibco products, such as buffers and growth factors, contact us at custommedia@lifetech.com.

Is it necessary to measure and adjust pH for the liquid media (classical or specialty) you sell, or are they already at the optimal pH?

No, you do not need to adjust the pH of the liquid media when you receive it. It should be in the correct pH range for use. After you add your supplements (if needed), you can check the pH again and adjust if necessary. However, usually it does not change significantly. It would be necessary to pH media that you make up yourself from a powder, with the exception of AGT media where pH and osmolality are auto-adjusted.

What quality water do you use in formulating your media?

Gibco cell culture media is formulated using water meeting all USP monograph requirements for Water For Injection.

How do you prepare cell culture media from powder or from concentrates?

You can find a protocol on our web site. In the main search box type “Media Preparation from Powder and Concentrates” and hit Enter or click Search. This should take you to a detailed protocol page.

How can I maintain an aseptic technique for cell culture?

Aseptic technique, designed to provide a barrier between the microrganisms in the environment and the sterile cell culture, depends upon a set of procedures to reduce the probability of contamination from these sources. The elements of aseptic technique are an aseptic work area, good personal hygiene, sterile (or sterile filtered) reagents and media, and aseptic handling. Go here to read more (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/aseptic-technique.html) about aseptic technique or view our checklist (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/aseptic-technique/aseptic-techniques-checklist.html).

What are the buffering conditions I should use for my CO2 incubator when culturing mammalian cells?

Most normal mammalian cell lines grow well at pH 7.4, and there is very little variability among different cell strains. While most researchers usually use 5-7% CO² in air, 4-10% CO² is common for most cell culture experiments. Read more about buffering conditions here (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-environment/ph-CO²-levels.html).

Do you have a comparison of dishes, culture plates, and flasks by surface area, seeding density, cells at confluency, and volumes of Versene solution, trypsin, and growth medium?

Please see the comparison chart here (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html).

Will improper storage of my tissue culture reagents affect the growth rate of my culture?

Yes. Store animal sera at -5 to -20 degrees C. Store media at 2 to 8 degrees C; use within recommended shelf life period. Store complete media (supplemented) at 2 to 8 degrees C, and for complete medium the recommended shelf life is 2 to 4 weeks. Additionally, minimize exposure of sera and media to light.

Where can I find media formulations?

Please use our media formulation search tool (https://www.thermofisher.com/search/results?docTypes=MediaFormulation&persona=DocSupport&linkIn=true&query=).

For Research Use or Further Manufacturing. Not for diagnostic use or direct administration into humans or animals.