Hybridoma

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Hybridoma

Postby gene26 » Jun 19 2016 1:19 pm

How is it possible to maintain cells under log phase. How do we know that they are under log phase?

I read in a book hybridoma cells should be under log-phase growth condition?

If we revive cells from -80C and incubated in fresh FBS 10% for 48hrs in CO2 incubator will they be under log phase?
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Re: Hybridoma

Postby sile88314 » Jun 20 2016 2:08 am

You may need to get the cell growth curve.
Inoculate a same amount of cells into several culture flasks with same standard. every 24h (up to you), take 1 flasks and count the number of cells. gather all the cell number data and make a curve, you may get a "S" Shaped curve, than you will know how long will your cells growth into log phase.
but it is not absolute, even a same cell ,the growth curve could be change, you should continuously passage the cells to maintain the cell in log phase, the time interval you do cell passage is up to the cell itself.
hope it can help.
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Re: Hybridoma

Postby gene26 » Jun 20 2016 6:27 pm

How do I inoculate same amount of cells? Is it like this, if *1ml has 1000 cells then I add 1ml in say 6ml FBS in n number 25cm2 flask and check the cell count after 24hrs. After 24hrs it would be 1000+ or maybe some cells may reach senescence and they could be ~1000 cells again? after 48hrs they would be 1000+ for sure. Now how do I know they are under log phase can u explain with an example.

*But if 1ml has 1000 cells that doesn't mean next 1ml will also have 1000 it could be more (1200) or less (750).

sile88314 wrote:you should continuously passage the cells to maintain the cell in log phase, the time interval you do cell passage is up to the cell itself
hope it can help.


continuously passage the cells? u mean I should inoculate some amount from previous medium in to fresh medium? and keep doing that, I don't get it.
Last edited by gene26 on Jun 21 2016 6:57 am, edited 1 time in total.
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Re: Hybridoma

Postby sile88314 » Jun 21 2016 1:34 am

sorry for that i'm not a native English speaker, so my expression may confuse you sometimes.
Generation of a growth curve can be useful in evaluating the growth characteristics of a cell line.From a growth curve, the lag time, population doubling time, and saturation
density can be determined.
Procedure:
1. Trypsinize and centrifuge the cells.
2. Resuspend the pellet in 5 mL of medium and count the cells(see Hemocytometer Counting).
3. Dilute the cell suspension in order to have an appropriate amount of medium and cells to achieve a seeding density of 2 x 103 cells per cm2 of surface area. Cell seeding
densities vary by cell line (you should choose an appropriate seeding density according to your cell, if you cell grow quickly, the density would be relatively low).
4. Mix well and seed the dishes/flasks with the appropriate amount of diluted cell suspension.
5. Count some of the leftover cell suspension in order to determine the actual seeding density
6. Put the plates in an incubator.
7. Count the duplicate plates every 24 hours (also up to your cells, if they grow too fast, this time should be shorten).
8. Plot the results on a log-linear scale. The population-doubling time can be determined by identifying a cell number along the exponential phase of the curve, tracing the
curve until that number has doubled, and calculating the time between the two.
u mean I should inoculate some amount from previous medium in to fresh medium? and keep doing that,

yes, I mean you should keep doing the cell passage, let the cell keep growing, and you may understand the "growing habit" of your cell and find a optimized time lag to do cell passage.
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Re: Hybridoma

Postby gene26 » Jun 21 2016 6:29 am

I'm a starter in cell culture

So lag phase has to be determined empirically then Eg: If I seeded 1*10^5 on day one after 48hrs it doubled say 2*10^5 (it means cells are in log phase?) then they increased to 3x10^2 after 72 hrs and maintained for next 24hrs it implies stationary phase? and then decreased (I don't know if this will be the case in practice I'm just assuming here).
Now to maintain under log phase, I should inoculate the cells from previous medium or revive them from -80C and change the medium before/within 48hrs and keep inoculating them within every 48hrs Is that correct? When cells are in log phase it means they adapted to the milieu and proliferate rapidly. so if we keep change the medium within every 48hrs with addition of few cells (inoculation) it is like they are trying to adapt each time and start to divide. So the cells divide in lag phase what is the difference between lag and log just the number right? obviously the number doubles overtime, so why does it matter if cells are in lag or log? what is the significance of maintaining them under log phase.

From your explanation cell seeding implies to determine how many cells we add thru dilution. What is cell at confluency? n number cells bound to ncm2 flask? in thermo website it was mentioned for 25cm2 HeLa cells has confluency 2.8 x 10^6 so when there are 2.8 x 10^6 cells it means that the whole area is saturated or maybe the flask might form a layer beyond monolayer? so we need to maintain the cells under 2.8 x 10^6? or else they stop proliferation? SP2 cells are immortal does it affect in this case? I guess cells will go beyond monolayer.
sile88314 wrote:if you cell grow quickly, the density would be relatively low

When I saw it under microscope they were so many cells (looks like it formed monolayer to me) after day 3. How is the density low when they grow quickly? if they are more cells they would get densely packed?
sile88314 wrote:5. Count some of the leftover cell suspension in order to determine the actual seeding density

this leftover cell suspension is from making seeds thru dilution in step 2
sile88314 wrote:2. Resuspend the pellet in 5 mL of medium and count the cells
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