Cell Culture Freezing Media

Following on from the last post in regards to freezing cell culture stocks, I thought it would interest you in knowing the type of freezing media I generally use for each stock type. For my Master and Submaster cell stocks, I generally use a freezing media composition of 10% DMSO + 90% FBS. In contrast, for the Working cell stocks, I would use 10% DMSO + cell culture media containing 10% FBS.

High Percentage Serum

As you may know, DMSO is typically used because it has low toxicity to mammalian cells and importantly, prevents the formation of disruptive ice crystals during the freezing process. I opt for the higher serum percentage for the Master and Submaster cell stocks because it contains less water compared to cell culture media + serum. As such, the high percentage serum in combination with DMSO makes for a “gentle” freezing media capable of preserving cell viability or enhancing recovery after thawing. This is particularly important if the cell stocks are going to stay stored in liquid nitrogen for very long periods (i.e. years).

Lower Percentage Serum

Regarding the Working cells, I generally opt for a lower percentage serum for freezing because the Working cell stocks are not meant for long-term storage as compared to the Master cells and because they have a higher turnover in terms of usage. There is nothing wrong with using 10% serum; cell viability can be just as good as if you had frozen your cells using 90% serum. The main difference I have noticed comes from long-term storage (e.g. 2+ years).

Another reason to use a lower percentage serum for the Working cells is cost. A high number of Working cell stocks are generally frozen down so if you are using 90% serum, it will get expensive.

But despite what I have said above, if you are using a cell type that is just bad to freeze-thaw (i.e. not very good at recovering quickly), use the higher percentage serum to freeze down your Working cell stocks.

Ways To Ensure Experimental Consistency #1

When reproducing experiments, it is important to keep experimental variation and human error to a minimum.

Cell-line Stocks

One way to ensure consistency is to keep track of the passage numbers of your cell-lines. When you order in new cells or are given cells from a collaborator, make a note of how many passages they have already been through.  Once this is done, proceed to create your own stocks. I have typically used the organization as set out below.

The Master cells are cells that you expand upon receipt. Freeze a high concentration (e.g. ~1x10⁷ cells/ml) of these down as your Master cell stocks and save an aliquot to continue growing for the Submaster cell stocks. For the Submaster stocks, you can freeze at a lower cell concentration (e.g. ~5x10⁶ cells/ml). After freezing down aliquots of your Submaster cell stocks, once again, save an aliquot for further expansion for your Working cell stocks. The Working cells can be frozen anywhere between 1-5x10⁶ cells/ml.

It is intended that the Working cells be used for x number of experiments before a new vial is thawed. It is only once you exhaust the Working stocks that you thaw out one vial of your Submaster cell stocks for expansion to create a new Working cell stock. From this you can see that it is only once your Submaster stocks are exhausted do you then thaw out one of your Master cells vials to repeat the entire process.

Keep a log book of your cell stocks and make sure that the numbers are regularly updated.

Online Databases – The GEO DataSet Browser

A great way of gathering some preliminary data is to use the GEO DataSet Browser on NCBI. NCBI has many useful resources and this database is one. Examples of its use include look at gene expression changes in:

* tissues stimulated with steroids, drugs, and other compounds;

* control vs diseased tissues;

* the effect of age;

* etc

Here is a link to the GEO DataSet Browser:

http://www.ncbi.nlm.nih.gov/sites/GDSbrowser/

For those of you not familiar with it, just type in your search term in the search box e.g. “alzheimers disease stages”. Select a GDS dataset. Scroll down to the bottom and you should see 3 tabs “Expression Profiles”, “Data Analysis Tools” and “Sample Subsets”. The “Expression Profiles” tab will take you to the profiles of all the genes screened.  When you click on the “Data Analysis Tools” tab a new section will appear below. Type in the name of your GOI in the search box and you will be able to search for gene expression changes in your GOI. The “Sample Subsets” tab provides information about the samples used.

Western Blotting – Why Are My Gels Not Setting?

This question comes up quite often. It can be frustrating having to work to a tight schedule and when for whatever reason your separating and/or stacking gel decides not to set, plans can be sent into a tailspin. 

Usually when a gel doesn’t set, you just start again, but you will be wondering why it didn’t set in the first place. Some common causes are obvious but some are less so.

Clean Your Plates

Those glass plates really need to be clean. If you have residue left over from the previous western run, it can impact on gel polymerization. The same goes for the combs, they need to be cleaned too.

Just before assembling your plates for pouring, spray and wipe (using lint-free tissues) the plates and combs with 70% ethanol.

APS/TEMED

APS and TEMED are important components of the gel mix as they are required for the polymerization of the acrylamide monomers. APS forms the free radicals needed for the polymerization process while TEMED stabilizes those free radicals to speed up the entire process.

APS goes off very easily. If you have made fresh APS and stored it at 4 degrees, it should be fine for about 1-2 weeks (depending on the frequency of use), provided you have not left it to sit at room temperature for extended periods. APS sitting at room temperature for hours should be thrown out.

Ideally, it is better to make fresh APS for each experiment. Just weigh out what you need for each experiment and reconstitute with water.

If despite using fresh APS you find that your gels are setting very slowly i.e. taking hours instead of minutes, you may want to consider using new TEMED.

Delta-Delta CT/Comparative CT Method Of Calculating Real-Time PCR Data Using Excel

For those who prefer to calculate their real-time PCR or qPCR data using the DDCT (also referred to as comparative CT) method but want to be able to evaluate the calculation of the data step by step, the following set of Excel formulae will come in handy. The Columns referred to correspond to the columns in the figure below.

Cell Formulae

Column B: Gene 1 CT values from your qPCR run

Column C: Gene 2 CT values from your qPCR run

Column D: Housekeeping gene CT values from your qPCR run

Column E: =B4-D4, =B5-D5, etc

Column F: =C4-D4, =C5-D5, etc

Column G: =AVERAGE(E4:E6)

Column H: =AVERAGE(F4:F6)

Column I: =E4-$G$4, =E5-$G$4, etc

Column J: =F4-$H$4, =F5-$H$4, etc

Column K: =POWER(2,-I4),=POWER(2,-I5), etc

Column L: =POWER(2,-J4), =POWER(2,-J5), etc

Column M: =AVERAGE(K4:K6), =AVERAGE(K7:K9), etc

Column N: =AVERAGE(L4:L6), =AVERAGE(L7:L9), etc

Column O: =STDEV(K4:K6), =STDEV(K7:K9),etc

Column P: =STDEV(L4:L6), =STDEV(L7:L9), etc

Column Q: =O4/SQRT(3), =O7/SQRT(3), etc

Column R: =P4/SQRT(3), =P7/SQRT(3), etc