7 replies to this topic

[madelingirly]

Dear Guys

I have some questions about cells grow in suspension, I will be very grateful for those who are willing to share their experience with me. As it is easier to understand steps for adherent cells more than suspension cells.

At first, the confluency in adherent cells determined by the cells cover the plate surface (70-80%) of plate
How is confluency determined for suspension cells ( I read some thing about cell number per ml medium) then it is consider confluent.
Please could some one confirm this data for me.


Secondly, shall i restart culturing of my cells from frozen stock in small (25ml flask) or 75 ml flask my stock conatins milion cells in 1 ml.

Thirdly, in adherent cells I just remove medium wash with saline like PBS, then add new medium in maintaining the cells.
Does maintaining suspension cells include only removing part of medium (of course will contain cells) and add new fresh medium.
The second thing how I can determine this part, (if I will culture it 75 ml flask).

Fourthly, at confluency, I read about 1:5 or 1:4 or 1:3 splitting (does this mean I take 10 ml of my cells for example and dilute it with 30 ml medium (for 1:3 splitting) in a new culture flask.

Finally, If I want to do test like viability test, and I have no centrifugation device for plates (lack of resources), what is the possible solutions,  I have this idea of  culturing the cells in 24- well plate for example, and take the content of each well and centrifugate it in microtube for example, then return it back to well in each step of experiment.

Finally, Thanks Guys for your priceless help and time
Best Regards

[bob1]

I have never used suspension cells, so I don't know the density at which they should be subcultured, which will depend on the cell line anyway

The best way to subculture is to take a proportion of the cells (taking a third of the volume is 1:3; a fifth, 1:5; etc.) and pellet them in a centrifuge (100-300 rcf for 5 min), then resuspend in fresh medium. to the appropriate volume and place in a  new flask.

A basic viability test is to take a sample of your cells and measure the proportion that stain blue with trypan blue (normal live/dead cell count).  Otherwise you could measure proliferation by a MTT or similar test.

[rhombus]

madelingirly, on 01 March 2012 - 08:38 PM, said:

Dear Guys

I have some questions about cells grow in suspension, I will be very grateful for those who are willing to share their experience with me. As it is easier to understand steps for adherent cells more than suspension cells.

At first, the confluency in adherent cells determined by the cells cover the plate surface (70-80%) of plate
How is confluency determined for suspension cells ( I read some thing about cell number per ml medium) then it is consider confluent.
Please could some one confirm this data for me.


Secondly, shall i restart culturing of my cells from frozen stock in small (25ml flask) or 75 ml flask my stock conatins milion cells in 1 ml.

Thirdly, in adherent cells I just remove medium wash with saline like PBS, then add new medium in maintaining the cells.
Does maintaining suspension cells include only removing part of medium (of course will contain cells) and add new fresh medium.
The second thing how I can determine this part, (if I will culture it 75 ml flask).

Fourthly, at confluency, I read about 1:5 or 1:4 or 1:3 splitting (does this mean I take 10 ml of my cells for example and dilute it with 30 ml medium (for 1:3 splitting) in a new culture flask.

Finally, If I want to do test like viability test, and I have no centrifugation device for plates (lack of resources), what is the possible solutions,  I have this idea of  culturing the cells in 24- well plate for example, and take the content of each well and centrifugate it in microtube for example, then return it back to well in each step of experiment.

Finally, Thanks Guys for your priceless help and time
Best Regards

Dear Madelingirly,

I will try and answer your questions in order:

a. The suspension cells I have grown include Sf9,Sf21, J774, RAW 267.4, THP-1, Jurkat, P388 etc. In all cases the cells are kept between 200,000 cells/ml and 800,000 cells/ml. Below and above those limits the cells struggle.

b. What you use for INITIATING your cells from frozen stock depends entirely on how well your frozen stocks recover from liquid nitrogen storage....AND this depends upon how well they have been frozen down in the first place. When I freeze cells down I always one week later check their viability and sterility. In that way it gives me a good idea on what flask to use later when I am using them for experimenting.

c. So what I do is use 80% of the growing cells for my experiments. The other 20% are centrifuged, resuspended in fresh media and then split accordingly into flasks.

d. Split ratio's then:-
1:5 means that 1 x 25cm TC flask will spilt into 5 .......that is if you do not use the original flask....if you keep the original falsk then you only need 4.
and so on for 1:4 and 1:3.
Because no trypsin is use to remove the cells from the plastic AND the cells are not growing on the TC surface....I do re-use flask's to save money. However the most important thing is to check the 0.2uM filter cap....this will not last for ever.

e. The only thing you can do is test the viability of the cell suspension at maximum concentration i.e. 800,000 cells/ml......if you are using a NEURBAUYER HAEMOCYTOMETER/TRYPAN BLUE EXCLUSION TEST there should be enough cells to do a reliable and reproducible count.


Hopefully some of this information will be useful

Kindest regards and good luck

Uncle Rhombus.

[madelingirly]

Dear

bob1

I am very grateful for your helpful comments
Best Regards

[madelingirly]

Dear Uncle
rhombus


I am very grateful for your priceless help and time.

But I cant understand something u have written
" 1 x 25cm TC flask will spilt into 5"
Does this mean if I am culturing in 75 cm TC flask, so I shall have 3 volumes (equivalent to 25 cm) each volume could be splitted
So, If I have 30 ml of medium in TC flask, Each 10 ml could be splitted ( if my ratio is 1:5) into 5 flasks,
So I shall take 2 ml of my flask and dilute it with 13 ml medium in a new TC flask and repeat it for 5 flasks.
So confluent flask can subculture upto 15 flasks in 1:5 splitting ration.
Please confirm if I understood it correctly.
Regarding my question about cell viability, I mean MTT assay for example, as there are steps for medium removal and additional of new reagents and a subsequent removal, so I was seeking the advice of any one who suffer from lack of machines suitable for TC plate centrifugation,
any thoughts or previous experience regarding MTT test or likewise viability test which requires further removal of medium or reagents from the cells growing in suspensions.

Finally, I am deeply indebted for your helpful comments

[rhombus]

madelingirly, on 05 March 2012 - 02:26 AM, said:

Dear Uncle
rhombus


I am very grateful for your priceless help and time.

But I cant understand something u have written
" 1 x 25cm TC flask will spilt into 5"
Does this mean if I am culturing in 75 cm TC flask, so I shall have 3 volumes (equivalent to 25 cm) each volume could be splitted
So, If I have 30 ml of medium in TC flask, Each 10 ml could be splitted ( if my ratio is 1:5) into 5 flasks,
So I shall take 2 ml of my flask and dilute it with 13 ml medium in a new TC flask and repeat it for 5 flasks.
So confluent flask can subculture upto 15 flasks in 1:5 splitting ration.
Please confirm if I understood it correctly.
Regarding my question about cell viability, I mean MTT assay for example, as there are steps for medium removal and additional of new reagents and a subsequent removal, so I was seeking the advice of any one who suffer from lack of machines suitable for TC plate centrifugation,
any thoughts or previous experience regarding MTT test or likewise viability test which requires further removal of medium or reagents from the cells growing in suspensions.

Finally, I am deeply indebted for your helpful comments

Dear madelingirly,

If you have a T75cm flask, then the flask will need to be split into 5 new flasks if you are adhering to a 1:5 split ratio.
If in your T75cm flask you have 30 ml of cell suspension, then 6mls of cell suspension would be used ti SEED each new T75cm flask (for a 1:5 split ratio

I do not do MTT assays so I cannot help with that.

Kindest regards

Uncle Rhombus

[bob1]

madelingirly, on 05 March 2012 - 02:26 AM, said:

Regarding my question about cell viability, I mean MTT assay for example, as there are steps for medium removal and additional of new reagents and a subsequent removal, so I was seeking the advice of any one who suffer from lack of machines suitable for TC plate centrifugation,
any thoughts or previous experience regarding MTT test or likewise viability test which requires further removal of medium or reagents from the cells growing in suspensions.

Finally, I am deeply indebted for your helpful comments

In your case a simple trypan blue exclusion test should work fine.  You won't need any centrifuge at all for this, just take a small aliquot of cells+medium from the suspension, add trypan blue (0.4%) at a 1:1 ratio (e.g. if you take 40 ul, add 40 ul trypan blue solution), leave for a minute, fill haemocytometer and count.

Regards MTT - I think there are MTT assays that can be done in the medium without removing anything.  Having a search on the web would be your best bet, there are quite a few options out there.

[madelingirly]

Thanks Guys for your help
I am very grateful for these priceless advice