specially important if you want to preserve viability of complement-sensible cells. Complement may attach to many cells and kill them, specially in the presence of Antibodies (that for sure will also be in your sera). In general, it is a good routine to inactivate sera by heating 30min at 56º (in a water bath)...

Good luck

Putting FBS in a water bath at 56C for 30 minutes will NOT inactivate it. The FBS within the bottle must be at 56C for 30 minutes and it will not even have reached that temperature at the end of 30 minutes.

Hi,

this is totally case-dependent. Various FBS batches will have different amounts of complement (which, among other factors is why virtually all suppliers offer test samples and allow reservation of specific batches) and different cell lines will show specific sensitivity towards those proteins, so you will have to check this for the specific use.

Generally, FBS complement contents are very low compared to those in newborn or adult sera, which is why fetal sera are generally preferred (in addition to the high content of growth factors).

I agree with Ron, proper heat inactivation will require the use of a reference vessel (filled with water), in which you monitor the actual temperature.

Generally, I'd recommend to try the serum without heat-inactivation first (or ideally in parallel), as it will in many cases also compromise the growth-promoting effect. Personally, I'm culturing lines where heat-inactivation is recommended without any negative impact.

Ri --

Have you ever tested it?

Have you ever heated inactivated bottles of FBS by using comparably sized water bottles with a thermometer in it like I have done dozens of times?

I guarantee that you will find that such a bottle starting at room or fridge temperature will not reach 56C in a standard lab sized water bath within 30 minutes.

To claim otherwise is "terribly wrong."

Ri --

Ri --

Ri --

This is what I wrote...

"Putting FBS in a water bath at 56C for 30 minutes will NOT inactivate it. The FBS within the bottle must be at 56C for 30 minutes and it will not even have reached that temperature at the end of 30 minutes."

If you have a disagreement with that statement, then please do all the courtesy of stating WHAT about it is "terribly wrong." Also, please note that Patrick Heinrich agreed with me when he wrote...

"I agree with Ron, proper heat inactivation will require the use of a reference vessel (filled with water), in which you monitor the actual temperature."

So what specifically is "terribly wrong" what I wrote?

I am trying to learn as well. I offered what I considered to be a legitimate contribution to the topic. All you did was say that I was "terribly wrong."

I am not the one trolling here.

What was "terribly wrong" with what I wrote? If something was wrong with my statement as you claimed, I would like to know what it was.

"Putting FBS in a water bath at 56C for 30 minutes will NOT inactivate it. The FBS within the bottle must be at 56C for 30 minutes and it will not even have reached that temperature at the end of 30 minutes."

I'm the one that made a contribution. Anyone is welcome to disagree with it, but since "there are people that are actually trying to learn here," they need to explain the basis of their disagreement...

"Putting FBS in a water bath at 56C for 30 minutes will NOT inactivate it. The FBS within the bottle must be at 56C for 30 minutes and it will not even have reached that temperature at the end of 30 minutes."

I have done FBS heat inactivations many times. I always have used a reference bottle with a thermometer in it. The water level of the water bath must reach the same level as that of the FBS in the bottles to ensure uniform heating of the bottles. The liquid in the reference bottle must be at the same starting temperature as the FBS bottles. When doing this, whether inactivating several bottles or even only one bottle, the temperature of the reference bottle definitely will not reach 56C in 30 minutes when using a standard laboratory water bath. Therefore heat inactivation is not accomplished by putting a bottle of serum into a 56C water bath only for 30 minutes.

Below is from the first source I found with a google search. No doubt there are others but this one will do...

Source: https://atlasbio.com/support/protocols/heat-inactivation/

Purpose The following protocol is suggested for the heat inactivation of bovine serum. Bovine serum is heated to 56± 2°C for 30± 2 minutes in a water-bath. Note: Our heat inactivation procedure may not be the same as the one your lab is using. Keep in mind that heat inactivation requires the serum to be at 56°C for 30 minutes. Prior to reaching 56°C, the serum is in a water bath at elevated temperatures for approximately 1 hour, the time it takes for the serum in the bottle to reach 56°C. This is very different than putting the serum in a 56°C water bath for 30 minutes, which is not heat inactivation by definition. If you ask us to perform heat inactivation we use a detailed, documented procedure and we are not responsible for inconsistencies in performance when compared with serum using a different procedure.

Here is a souce for another inactivation protocol...

http://www.serumsourceintl.com/pdf/heat_inactivation_instructions.pdf

Heat transfer is not instantaneous. It takes more than 30 minutes for a room temperature bottle of serum to reach 56C in a standard sized lab water bath. I made this statement based upon years of experience doing heat inactivations and I have supported it with a reference.

I myself am a credible source and I have written based upon my direct knowledge on this matter. There is nothing indicating any lack of legitimacy for those websites (how can an FBS vendor not be legitimate?) and they describe exactly what I have experienced.

However, regarding legitimacy, I follow requisite community standards here using my full name and affiliation.

My answer remained on topic. Any legitimate disagreement with it should be explained.

"Putting FBS in a water bath at 56C for 30 minutes will NOT inactivate it. The FBS within the bottle must be at 56C for 30 minutes and it will not even have reached that temperature at the end of 30 minutes."

I addressed the question here.

You simply said I was wrong with NO explanation.

I have provided sources to back up my answer. You said they were illegitimate. They were not.

I have provided my full name and affiliation as required by the community standards here.

You hide behind two letters, providing neither your name nor affiliation.

I am not the one trolling here.

Using a water bath equilibrated to 56C overnight, I put two room temperature 500ml bottles into the water bath. One was a reference bottle with a thermometer in it.

Time 0': water bath 56.0C bottle 22C

Time 10': water bath 53.0C bottle 43C

Time 20': water bath 52.9C bottle 49C

Time 30': water bath 54.0C bottle 51C

Time 40": water bath 55.3C bottle 53C

This shows exactly why you cannot simply put a bottle of FBS into a water bath for 30 minutes and expect it to be heat inactivated.