Trypsin-EDTA (0.05%), phenol red, 500 mL - FAQs

解冻后的胰酶可在冰箱中放置多长时间？

通常情况下，胰酶可在冰箱中保存一周。不过，这一时间长度也随着这一周内的加热次数和使用频率而变化。同时，它也与细胞粘附的紧密程度有关，胰酶在非冰冻条件下每天都会丧失一定的活性。

Gibco TrypLE试剂与胰酶相比优势在于？

TrypLE试剂不含动物源性与人源成份，纯度极高且对细胞的作用十分温和。该产品无需使用胰酶抑制剂对其进行终止。在含血清和不含血清的培养条件下，TrypLE试剂都是解离各类贴壁培养的哺乳动物细胞系的理想之选，能够直接做为胰酶替代品而应用于您当前的实验方案。

你们提供哪些用于解离细胞的试剂？这些产品之间的主要区别是什么？

请通过此选择列表(https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin.html)来比较我们的细胞解离试剂。

你们是否提供不含酚红的培养基（无酚红培养基）？

是的，请访问此页面(https://tools.thermofisher.com/content/sfs/brochures/Phenol_RedFree_Media_Glance.pdf)浏览我们的无酚红培养基页面。

How do you isolate cells from tissue?

Dissociation of cells from Primary Tissue

TRYPSIN
1. After dissecting off unusable tissue, mince the remaining tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces by resuspending in a balanced salt solution without calcium and magnesium. Allow the tissue pieces to settle, and remove the supernatant. Repeat the wash 2 or 3 times.
2. Place the container with the tissue pieces on ice, and remove any remaining supernatant. Add 0.25% trypsin in a balanced salt solution without calcium or magnesium (1 ml of trypsin for every 100 mg of tissue).
3. Incubate at 4°C for 6 to 18 h to maximize penetration of the enzyme with little trypsin activity.
4. Decant and discard the trypsin from the tissue pieces. Incubate the tissue pieces with residual trypsin at 37°C for 20 to 30 min.
5. Add warm, complete media to the tissue pieces and gently disperse the tissue by pipetting. If using a serum-free medium, also add soybean trypsin inhibitor.
6. Filter the cell suspension through sterile, stainless steel mesh (100 to 200 µm) to completely disperse any remaining tissue. Count and seed the cells for culture.

COLLAGENASE
1. Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces several times with Hanks' Balanced Salt Solution (HBSS).
2. Add collagenase (50 to 200 U/ml in HBSS).
3. Incubate at 37°C for 4 to 18 h. Addition of 3 mM CaCl2 increases the efficiency of dissociation.
4. Filter the cell suspension through a sterile stainless steel or nylon mesh to separate the dispersed cells and tissue fragments from the larger pieces. Fresh collagenase can be added to the fragments if further disaggregation is required.
5. Wash suspension several times by centrifugation in HBSS.
6. Resuspend the pellet in culture medium. Count and seed the cells for culture.

DISPASE
1. Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces several times in a calcium and magnesium-free balanced salt solution.
2. Add dispase (0.6 to 2.4 U/ml in calcium and magnesium-free balanced salt solution).
3. Incubate at 37°C for 20 min to several hours.
4. Filter the cell suspension through a sterile, stainless steel or nylon mesh to separate the dispersed cells and tissue fragments from the larger pieces. Fresh dispase can be added to the fragments if further disaggregation is required.
5. Wash suspension several times by centrifugation in the balanced salt solution.
6. Resuspend the pellet in culture medium. Count and seed the cells for culture.

REFERENCE:
Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.

Does Diploid Growth Serum-Reduced Medium (SRM) contain L-glutamine?

The basal Diploid Growth Serum-Reduced Medium already contains 6 mM L-glutamine.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Do I have to adjust my infection parameters when using Diploid Production Serum-Free Medium (SFM)?

We recommend evaluating performance with Diploid Production Serum-Free Medium using current conditions, however, multiplicity of infection, time of infection, and time of harvest may be different than with conventional media. Closely monitor cells for cytopathic effect and evaluate viral titers at multiple time points.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Can Diploid Growth Serum-Reduced Medium (SRM) be supplemented with different types of serum?

Diploid Growth Serum-Reduced Medium can be supplemented with Fetal Bovine Serum, Newborn Calf Serum, Donor Bovine Serum with Iron, or Bovine Serum.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Which cell lines can I culture in Diploid Growth Serum-Reduced Medium (SRM)?

Diploid Growth Serum-Reduced Medium has been evaluated with fibroblast cell lines, including MRC-5, WI-38, IMR-90, BS-2, and CEF cells.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

How long can I use Diploid Growth Serum-Reduced Medium (SRM) supplemented with Diploid Growth Supplement?

Once supplemented, the complete Diploid Growth Serum-Reduced Medium must be used within 4 weeks.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Can I freeze or thaw my cells in Diploid Growth Serum-Reduced Medium (SRM)?

Most diploid cell lines can be frozen and thawed in Diploid Growth Serum-Reduced Medium. Consult the Diploid Growth Serum Reduced Medium User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0019015_DiploidGrowthSerumReducedMedium_UG.pdf) for additional information.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

How do I adapt my cells to Diploid Growth Serum-Reduced Medium (SRM)?

Cells growing in medium with serum can be transferred directly to Diploid Growth Serum-Reduced Medium without adaptation. Continue to monitor and passage cells for 2-3 passages until consistent growth is achieved. Cultures may benefit from supplementation with 1-2% serum.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

How long can I keep trypsin in the refrigerator after thawing?

Generally speaking, trypsin can be stored refrigerated for up to a week. However, this may vary depending on how often it is warmed up and used during that week. It may also depend on how tightly the cells are attached, because there will be some loss of activity each day the trypsin is not frozen.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Do you have a non-animal-origin enzyme for cell dissociation?

Yes, the TrypLE products contain rProtease, a non-animal, trypsin-like enzyme used for the dissociation of attachment dependent cell lines. TrypLE enzyme has demonstrated the ability to dissociate cells cultured both in serum-free and serum-supplemented systems. The catalog numbers for TrypLE Select are 12563-011 (100 mL) and 12563-029 (500 mL). Some example catalog numbers for TrypLE Express include 12604-013 without Phenol red (100 mL) and 12605-010 with Phenol red (100 mL). Both formulations are also available in additional sizes. All these products are animal origin-free.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

What is the difference between TrypLE Express Enzyme, TrypLE Select Enzyme, and CTS TrypLE Select Enzyme?

All the above products contain rProtease, a non-animal, trypsin-like enzyme formulation used for the dissociation of attachment-dependent cell lines. This alternative for porcine trypsin is a recombinant enzyme derived from microbial fermentation.

The TrpLE Express, TrypLE Select, and CTS TrypLE Select products differ in their production and testing methods and in the documentation provided.

TrypLE Select is for the industrial market and has release-testing both for endotoxins and enzyme activity. This material also has a Drug Master File on record. It is manufactured in AOF-dedicated equipment and is intended for Research Use/Further Cell Culture Manufacturing.

TrypLE Express is manufactured without use of dedicated equipment and is intended for Research Use Only. Not for use in diagnostic procedures.

CTS TrypLE Select is manufactured under scalable cGMP conditions using dedicated equipment and is designed for clinical applications. This product has a Drug Master File on record and is intended for Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. Not intended for direct administration into humans or animals.

To read more about TrypLE and cell release times using this reagent, please go here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express.html#1).

Find additional tips, troubleshooting help, and resources within ourMammalian Cell Culture Basics Support Center.

What are the benefits of Gibco TrypLE reagent over trypsin?

TrypLE reagent is free of animal- and human-derived components, producing an exceptionally pure reagent that is gentle on cells. Inactivation with trypsin inhibitors is not required. TrypLE reagent is ideal for dissociating a variety of attachment-dependent mammalian cell lines both in serum and in serum-free conditions, and can be directly substituted for trypsin in your current protocol. TrypLE reagent is room-temperature stable and ready to use when you need it. TrypLE cell dissociation reagents remain stable for 24 months at room temperature, making storage and handling easier and more convenient.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

What reagents do you offer for cell dissociation, and what are the main differences between them?

Please use this selection chart that compares our cell dissociation reagents (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin.html).

Find additional tips, troubleshooting help, and resources within ourMammalian Cell Culture Basics Support Center.

Do you offer media without phenol red (phenol red-free media)?

Yes, please visit this page for a list of our phenol red-free media (http://tools.thermofisher.com/content/sfs/brochures/Phenol_RedFree_Media_Glance.pdf).

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.