New to Cell Culture and need some basic information - help needed (Apr/15/2008 )

Hey guys,

I have some questions regarding basic cell culture. I am currently using two different cell lines namely AGS and MKN74 cell lines. I am totally new to cell culture and my supervisor dont really teach me from scratch.

I have few questions

Let's say I have cells on 75 ml flask

For splitting cells

1) How do you calculate the confluency based on your naked eye from microscope?
2) How much cells do I need to split it? Is it 1:3 dilution or 1:5 dilution?
3) How often do you split your cells and is it same for most cell lines?

For subculture cells for infection

1) He, my supervisor told me to use 80 to 90% confluency. What does he mean by that? I just assume 1:2 dilution and the next day I can get roughly 80 to 90% confluency.
2) For infection, how many cells should I put in say in each well in 6 wells plate?
3) Do I really need tryphan blue and a haemocytometer to count the cells before proceed or I can just do some rough calculation?

I think I sound confused but I really am.

Thanks for any input.

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First of all check the cell lines in ATCC or the company`s webpage you get the cells from. There must be info about splitting, freezing, medium etc.

The 1. answer would be a bit subjective. When I look at the dish, 50% confluency means that the cellfree area should be the half of the whole dish area. 90-95% confluency means that cells cover the whole dish area but only some wholes between cells are visible. The way you plate is very important for culturing cells. The cells need to be evenly distributed on the dish

2. You can experiment a bit. Make different dilutions of your cells and also count them. That will give you a roughly idea. At which dilution do your cells get ready for the next passage, how many cells do you plate (for flask and dish)? How many cells do you have when the dish/flask get ready for splitting?

The cell number differs from cell line to cell line.

3. I split mines every 2-3 days (Monday, Wednesday, Friday, Monday...). It depends on cell line and how nice your cells are growing (1:3-1:8). It varies as I said.

1. What kind of virus? 80-90% sounds right. It depends again how fast your cells grow. You should know them by heart. Maybe 1:3 would be also okay (dunno)

2. ??

3. I count mines on Neubauer count chamber for transfection. For infections I just plated them at the right dilution (you get the feeling for that )

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thanks zek!

I roughly know what is going on since my first cell culture failed miserably because of the cells condition.

I am beginning to know how and where to look at under the microscope. I was so lost during the first few days.

I roughly know I guess but wish me luck. Thanks

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Good luck

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Hey..none of us walked into a tissue culture room and knew exactly what we were doing and how to take care of a specific cell line from day one. I don't know about anyone else, but it took me awhile to get the hang of the whole thing. Lord knows I contaminated tons of cells in the beginning and couldn't hit the proper confluency to save my life. Give yourself some time to get familiar with your cells and get to know them. Hang in there and before you know it, you'll be an expert! Good luck.

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Hmm... looks like you're working on cell lines over there...

i found on internet once, a pdf file with types of cells and the cell density they recommend to grow different flask/wells.
different cells has diffferent size, so different no of cells can be seeded into flask.
I'll try dig up and if possible i'll e-mail u.

GOOD LUCK!!!

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practice and observation i'm afraid

cell culture changes from cell type, P no, your availability and general mood

the confluency is a matter of personal judgement improved over time(look at some pics online - of different cell types - to get an idea)

the split depends on how well the cells grow and when you want to do the exp (less harsh split = sooner confluency)

you split when they reach 90-100% to stop them dying (falling off etc) - some take days, some take weeks

cell culture tends to live its own life with you turning up every now and again to tweak it

i'm supprised rhombus hasn't chipped in (he's not square - he's rhomboid!)

anyway

good luck

dom

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Dear Dom,

Your doing just fine, you almost sound like an expert !!!!!!!
I do switch off when the first line of the thread states " my supervisor don't really teach me from scratch"

As Al Murray (the pub landlord) would say " shame on him, shame on him, shame on him"

What's a supervisor supposed to do then?

All timjim's questions are laughably easy to sort out..... I cover this in the first hour of training in my Institute.

Keep up the good work Dom, you will get there in the end

Rhombus (rather more round than Rhomboid these days).

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ALMOST!!!

huh

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Dear Rhombus,

I know it is easy, but unfortunately, I am not getting any training. Just some rough estimation on how to grow cell culture etc. I am sure there are alot of other people who are expert in cell culture.

I am trying to learn as well. I am trying this little experiment on my own to grow cells in different dilution and observe under the microscope. The confluency here and there, quite tough to estimate sometimes.

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