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General Cell lines questions

Handling of cells upon arrival

What are the shipping conditions of the cells and is it critical to thaw them upon arrival?

In general, InvivoGen’s cells are shipped on dry-ice and since dry-ice is not nearly as cold as liquid nitrogen, thawing of the cells technically begins during transport. Thus, we recommend a full thaw upon receipt to ensure the best recovery results.
NOTE: this is not applicable in Asia as cell lines are sent at room temperature using our specially designed flask.

Can you provide any tips or advice regarding the initial cell culture procedure?

The most important step is the thawing procedure upon receipt of the cells. Do not put your cells at -80°C or in liquid nitrogen as this may damage the cells. You must propagate the cells immediately upon receipt.

I’m having trouble growing my cells (HEK and THP1), do you have any tips for my initial culture?

Below are a few tips we recommend if you encounter any issues during initial culture:
• For the first 2-3 passages, grow cells in media containing 20% FBS and no antibiotics.
• Do not allow cells to reach 100% confluency. Please check the cells as regularly as possible.
• The cells should not be grown in 20% FBS for too long. Use media with 10% FBS after 2 or 3 passages.
• When making frozen stocks, continue growing additional cultures in case there is a problem with the frozen stock.

If you are working with THP1 cells in particular, here are a few additional tips:
• THP1 cells need to be passaged between 3 x 105 and 7 x 105 cells per mL (we recommend 5 x 105 cells per ml). Below this, your cells will take a long time to grow and above 2 x 106 cells per ml you may have toxicity.
• Between each passage, do not centrifuge the cells. THP1 cells grow better in conditioned media, therefore when passaging cells, leave 1 – 2 ml of the old media and add fresh media on top. Do not leave more than 50% of conditioned media as the cells will not have enough nutrients to properly grow.

Why do you recommend waiting for 2-3 passages before adding selective antibiotics?

After thawing, the cells can be more sensitive to selective antibiotics due to the initial low levels of resistance markers. Therefore, it is recommended to wait for 2 - 3 passages before adding the selective antibiotics in order to avoid further stress to the cells during the first couple of passages.

Cell culture media

What serum is recommended for the culture of InvivoGen’s cells?

It is important to choose an endotoxin-free serum for the culture of our reporter cell lines. InvivoGen systematically asks for the Certificate of Analysis of different batches of serum to ensure a low level of endotoxin as to not activate the NFkB pathway.

What concentration of Pen/Strep should be added to the media?

100 U/ml Penicillin and 100 µg/ml Streptomycin

Is it a problem if the growth medium used for the cells does not contain the same concentration of L-glutamine as stated on the Technical Data Sheet?

No, this shouldn’t be a problem as long as there is at least 2 mM L-Glutamine in the medium. Most DMEM formulations already contain 4mM of L-Glutamine. However, if you need to add L-Glutamine to your medium you will only need to add 2 mM for the cells to grow properly.

Can I use Glutamine or GlutaMax?

Both Glutamine and GlutaMax have been tested in-house and can be used, with no difference in the cells noted.

Why do you recommend using heat-inactivated serum?

Many of InvivoGen’s cell lines are engineered with a SEAP (Secreted Embryonic Alkaline Phosphatase) reporter system, and FBS, in many cases, contains traces of alkaline phosphatase (AP) that interferes with the test results. Therefore, to avoid background noise we recommend working with inactivated serum with all of our cells.

Do you have a protocol for heat-inactivating FBS?

In house we inactivate serum by heating at 56°C for 30 minutes. It is important to wait for the water bath to reach the 56°C before placing the tube of serum in to ensure the serum is fully inactivated.

Does the freezing medium have to contain heat-inactivated FBS?

In house we always use heat-inactivated serum for the freezing medium, growth medium and test medium. However, please note that it is not a problem if you wish to use non-heat-inactivated in the freezing medium.

Cell line culture

My HEK cells are not properly attaching to the bottom of the flask. What type of plastic equipment do you recommend?

When the HEK-Blue™ cells are non-adherent, either they were diluted too harshly at the start or they have grown over-confluent in a small flask and suffocated.
To avoid this in the future:
• Change the medium and seed the cells at a density of approximately 1.5 x 106 cells/mL in a T25 flask.
• Wash the cells before putting them into a new flask. Sometimes when the cells are non-adherent, it is due to the clustering of both live and dead cells. Therefore, this will get rid of any remaining DMSO which could affect the adhesion of the cells to the flask.
• Use medium with 20% FBS.
• The use of CellBIND flasks can sometimes help to increase attachment and growth of the cells (however CellBIND flasks are not required in the normal protocol)

My THP1 cells have a doubling time of about a week. Is that expected?

It isn’t normal for your cells to divide only once a week. Depending on the clone, they usually have a doubling time of 24 – 72 hours. THP1 cells must be cultured at quite a high concentration (at least 5 x 105 cells/ml). Please note that our R&D teams have noticed that THP1 cells often die when they are diluted too harshly. Also, it may help to not add Normocin™ while waiting for improvement to their growth.

What do I do if THP1 cells are clumping but are not dead?

It is not a problem if the cells are clumping, as long as they are growing fine and have normal morphology. You may want to centrifuge the cells to get rid of the dead cells as sometimes this is the reason for clumping. Please note that we recommend homogenizing the cells before performing your assays.

How do I separate live cells from dead cells?

We recommend to centrifuge at 120 G for 10 minutes.

Assays

Can you provide tips to reduce the background noise in HEK-Blue™ cells?

To limit the activation of NFκB before stimulation (lower background):
• Use healthy cells that have been passaged at least 24 hours before the assay
• Do not allow cells to be greater than 80 % confluent
• Use pre-warmed PBS to rinse your cells
• Use heat-inactivated FBS (to eliminate residual alkaline phosphatase in the serum)
• Do not centrifuge the cells prior to stimulation
• Do not use trypsin (gently pipet up and down to detach cells)
• Do not use an excessive number of cells per well
(approximately 50 000 cells/well as recommended on the TDS).

Why do you recommend not add Normocin™ to the test medium and can selective antibiotics be added to the test Medium?

We recommend to not use Normocin™ in the test medium with all of our cell lines as it is better to reduce the number of potential interfering agents in the medium when performing the assay. The same goes for the use of selective antibiotics.

Can we expect similar results between assays when using the same number of cells and concentration of ligand, and the only difference being the passage number of the cells?

It is hard to estimate the deviation factor regarding cell passage number. However, we note very little difference in our experimental results, with no more than the slight variations you expect due to handling errors.

Can InvivoGen’s reporter cell lines be used in other assays, such as ELISA and Western blotting?

Yes, InvivoGen’s reporter cell lines can be used in ELISA and Western Blots. However, please note that we do not sell them for this purpose.

Can InvivoGen’s reporter cells be used for quantitative measurements?

They are semi-quantitative tests. However, they can be used for quantitative measurements by making a standard curve using a positive control.

Is it possible to freeze the supernatant after stimulation to read/detect the production of SEAP at a later date?

If you cannot run the detection assays immediately, you can store supernatant samples for up to a week at 4°C. Supernatant samples are active for a longer time when kept at -20°C. However, in this case we would recommend supplementing the test medium with 20% FBS to protect the SEAP as much as possible during the freezing process. Do not repeat freeze/thaw cycles.

Should the cells be pre-treated with the inhibitor or should the ligand be co-incubated with the inhibitor?

It depends on the inhibitor. If the inhibitor blocks a receptor or an element in the signaling pathway, we recommend pre-incubating the inhibitor with the cells for at least 30 minutes. If the inhibitor is binding or blocking the ligand (i.e. an antibody against a specific cytokine) then it is best to pre-incubate the inhibitor with the ligand prior to adding to the cells.

For how long prior to stimulation should the cells be seeded?

In house the cells are seeded at the same time as adding the ligand (ligand first).

Frozen stock preparation

What freezing medium should be used for adherent cells?

For adherent cells, we recommend DMEM, 20% (v/v) fetal bovine serum (FBS), and 10% (v/v) DMSO.

What freezing medium should be used for suspension cells?

For suspension cells, we recommend fetal bovine serum (FBS) and 10% DMSO.

What to do if the freezing step is not successful?

We highly recommend keeping a flask of cells running until you begin thawing your frozen stock in case something has gone wrong during the freezing step.

Reporter system

Does the production of SEAP require activation of both NFκB and AP-1 transcription factors, or is the activation of just one enough to get the signal?

Our reporter cells require the activation of just one transcription factor (NFκB and/or AP-1) to produce a signal.

From what species is the Lucia™ luciferase from?

InvivoGen’s Lucia™ luciferase is completely synthetic and is not related to any natural luciferase.

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