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User Guide: CTS Immune Cell SR Manual / Product Insert

  • Version: 1.0
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What are the storage conditions for CTS Immune Cell Serum Replacament (SR)? Upon thawing, can I aliquot and refreeze at -20 degrees C? Product FAQ

Answer

After completely thawing the initial product, it can be aliquoted and refrozen for another usage. The thawed aliquots are stable for 4 weeks when stored at 2-8 degrees C.

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

Answer Id: E17446

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Why do cells grow in lines when I am using glass Nunc Lab-Tek Chamber Slide products? How can I prevent this? Product FAQ

Answer

The following are possible causes and solutions:
- The cells could be stressed due to media substitution, especially from serum-free formulations, or other changes to the culture environment such as in temperature, humidity, and CO2 percentage. If possible, always plate cells in serum-containing medium. Switching cells to a serum-free media should be done gradually.
- The cells could be plated at very low densities. Most cell lines have a minimal platting density below which normal cell proliferation cannot occur, and therefore should be avoided.
- The cells could be poorly adherent. Treatment of cells that may reduce adhesion and therefore, should not be done immediately after plating, but after the cells have resumed exponential growth.

Answer Id: E17717

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What are the benefits of Gibco TrypLE reagent over trypsin? Product FAQ

Answer

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.

Answer Id: E11901

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My cells were not viable upon thaw. What happened? Product FAQ

Answer

Here are possible causes and solutions:

-Cells were not placed immediately either into culture or in liquid nitrogen. Cells are shipped overnight on dry ice, but cannot be left longer on dry ice, even for an additional day.
-The recommended medium/serum was not used. Cells have been FACS sorted and originate typically from a single cell and so are more sensitive to media and components. Please ensure that no substitutions are made.
-The DMSO was not removed from the medium. Please ensure that the DMSO is removed with a medium exchange as described in the protocol.

Find additional tips, troubleshooting help, and resources within our Drug Discovery & Development Support Center.

Answer Id: E16551

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Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute. Citations & References

  • Authors: Bernardo ME, Avanzini MA, Perotti C, Cometa AM, Moretta A, Lenta E, Del Fante C, Novara F, de Silvestri A, Amendola G, Zuffardi O, Maccario R, Locatelli F
  • Journal: J Cell Physiol
  • PubMed ID: 17187344
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In which protocols would you recommend substituting B-27 Plus Supplement and Neurobasal Plus Medium for classic B-27 and neurobasal products? Product FAQ

Answer

We recommend using B-27 Plus Supplement and Neurobasal Plus Medium instead of the regular versions of B-27 Supplement and Neurobasal Medium products (not specialty versions like minus Insulin, minus Vitamin-A or Neurobasal-A) in protocols used for:

- Maintenance of primary rat, mouse and human PSC-derived and fetal-derived neurons
- Differentiation of human PSC-derived and fetal-derived neural stem cells (NSCs) to neurons

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

Answer Id: E15878

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Application Note: Closed-system media formulations and culture systems for cell therapy manufacturing Product Literature

Can I expand your cells and re-freeze them? If so, how? Product FAQ

Answer

When either Gibco or Invitrogen cryopreserved or proliferating cultures are purchased from us, they may be expanded and cryopreserved again. However, the cryopreservation process may result in altered growth performance of the cells. The following protocol provides a basic guideline for the cryopreservation of cells using Synth-a-Freeze medium, a defined, protein-free cryopreservation medium available from us.

Please note: Due to differences in cryopreservation equipment and individual techniques, we cannot guarantee that cells cryopreserved using this protocol will be viable upon recovery from cryopreservation, and we do not provide a warranty for cells cryopreserved in an investigator's laboratory.

1. Thaw Synth-a-Freeze medium in a 37 degrees C water bath or overnight at 4 degrees C.
2. If thawed in a water bath, do not exceed 37 degrees C and do not leave the product at 37 degrees C for an extended period of time.
3. Synth-a-Freeze medium should be equilibrated to 4 degrees C prior to use. For optimal results, the use of a controlled-rate freezer is recommended. In the absence of a controlled-rate freezer, a cell cryopreservation container (e.g., Thermo Scientific Mr Frosty container) may be useful.
4. If enzymatic agents are used to remove the cells from a culture surface, resuspend the cells in a solution that will neutralize the effects of the enzyme.
5. Pellet the cells by centrifugation.
6. After removing the supernatant, resuspend the cell pellet in cold Synth-a-Freeze medium at a concentration of 5 x 10E5 to 3 x 10E6 cells/mL.
7. Distribute the cell suspension in an appropriate number of cryopreservation vials.
8. Cool the vials of cells to 4 degrees C as quickly as possible.
9. If using a controlled-rate freezer: freeze the material by reducing the temperature 1degrees C per minute until the temperature reaches -40 degrees C. Then reduce the temperature at a rate of 2 degrees C per minute until the temperature reaches approximately -90 degrees C.
10. If using a cell cryopreservation container: Prepare the container according to the manufacturer's instructions.
For best results we recommend transferring the vials to the vapor phase of a liquid nitrogen storage facility as soon as possible after the cells have reached -80 degrees C.

As a substitute for Synth-a-Freeze medium, the recommended basal medium for the cell type being cryopreserved, supplemented with 10% fetal bovine serum (FBS) and 10% DMSO, may be used. Please note that Synth-a-Freeze medium is NOT recommended for the cryopreservation of human epidermal melanocytes.

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

Answer Id: E11967

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

Answer

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

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

Answer Id: E11918

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I suspect mycoplasma is affecting the growth rate of my culture. How can I test for it? Product FAQ

Answer

There are several options. Our first recommendation is to use the Invitrogen MycoFluor Mycoplasma Detection Kit.

Mycoplasmas are small, self-replicating prokaryotes (0.3 - 0.8 mm diameter), that lack a cell wall and have the ability to adsorb onto host cells. Mycoplasma is one of the most serious forms of cryptic contamination and its presence is not detected unless appropriate tests are made or until some aspect of cell behavior is noticed to have changed. Between 15 and 50% of cell lines submitted to cell banks are contaminated with mycoplasma. Mycoplasma spreads readily among cell lines via reagents and media, the operator and the work surface.

The presence of mycoplasma may invalidate the results obtained with that culture. The presence of mycoplasma-infected cultures can result in the shut-down of the entire laboratory until the infection can be eliminated, whereupon complete restocking is required. The origin of contamination is usually traced to mycoplasma present in animal (bovine) serum or to human oral mycoplasma transferred by droplet infection during cell culture. The simplest test for the detection of mycoplasma in cultures is the use of a fluorescent dye which binds directly to DNA causing fluorescence (e.g. Hoechst 33258) which can be seen by fluorescence microscopy. Mycoplasma positive cells will show intense fluorescent spots on the plasma membranes or show filaments which may be absorbed onto the cells. Uncontaminated cells show only brightly fluorescent cell nuclei. The technique is rapid (less than 30 minutes), but requires heavy contamination (10E6 mycoplasma/ml) to produce a clear positive result. If however, the suspect cells are co-incubated for 2-4 days with an "indicator" cell line (such as 3T3) which is particularly suitable for demonstration of positive staining, then sensitivity can be substantially increased. Microbiological culture techniques are available that operate at a greater sensitivity, but it can take up to 21 days to obtain a result, a positive control is needed, and the result may require expert interpretation.

A variety of PCR-based methods are available, some of which have been utilized as commercially available detection kits. It is recommended to use a combination of DNA staining and a PCR-based method once every 3 months for all growing cultures in the laboratory and for every new cell line as it enters the laboratory. In addition, all Master and Working Cell Banks should be tested at the time of freezing. Quality control and good working practice will reduce potential problems. It is important that frozen stocks are created immediately after testing and re-tested before distribution. If cells are cultured for more than 3 months after testing, they should be re-tested. Regulatory bodies now insist that cell cultures used for the production of reagents for diagnostic kits or therapeutic agents are free from mycoplasma infection. Also, some scientific journals have the policy of requiring statements from authors that the culture work reported in those journals is carried out with mycoplasma-free cells. Normally, when contamination with mycoplasma is apparent, the recommendation would be to discard the cultures and start again. If necessary, and only if the contamination is not extensive, then it is often possible to rescue the cells by treatment with one of the commercially available antibiotics. This must only be considered for a remedial action, not as a routine supplement to growth media (and thereby a substitute for good cell culture practice).

Answer Id: E3983

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

Answer

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.

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

Answer Id: E11919

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Characterization and transplantation of induced megakaryocytes from hematopoietic stem cells for rapid platelet recovery by a two-step serum-free procedure Citations & References

  • Authors: Chen, TW; Hwang, SM; Chu, IM; Hsu, SC; Hsieh, TB; Yao, CL
  • Journal: EXPERIMENTAL HEMATOLOGY

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