4 replies to this topic

[Wek]

Could anyone give me brief explanation of the process in replacing a vial of cells when you plate it?
Do you grow the vial on a plate to ~80% confluency, trypsinize the cells, deactivate trypsin with medium, pellet the cells, resuspend cells in small volume (200ul) of medium, remove a small amount (~50ul) of cells for freezing and plate the remaining cells?

Also, how does passage numbering work? When exactly does it increase?

Edited by Wek, 16 December 2012 - 02:22 PM.

[science noob]

Wek, on 16 December 2012 - 02:21 PM, said:

Could anyone give me brief explanation of the process in replacing a vial of cells when you plate it?
Do you grow the vial on a plate to ~80% confluency, trypsinize the cells, deactivate trypsin with medium, pellet the cells, resuspend cells in small volume (200ul) of medium, remove a small amount (~50ul) of cells for freezing and plate the remaining cells?

Also, how does passage numbering work? When exactly does it increase?

What do you mean "replacing a vial of cells"?
Yup, your procedure sounds right.  You can choose to passage the cells after trypsinisation or you can freeze them down in DMSO.  Some people freeze cells in DMSO and media but we use DMSO diluted in serum (10% DMSO final concentration) and slow freeze the cells.

And your question about 'passage': https://www.google.c...iw=1366&bih=635

Our lab (*and most scientists) define 1 passage or P1 as when cells leave a monolayer culture flask/plate (via trypsinisation) and plated onto a fresh new flask - this is ONE passage.

[Wek]

science noob, on 16 December 2012 - 06:38 PM, said:

Wek, on 16 December 2012 - 02:21 PM, said:

Could anyone give me brief explanation of the process in replacing a vial of cells when you plate it?
Do you grow the vial on a plate to ~80% confluency, trypsinize the cells, deactivate trypsin with medium, pellet the cells, resuspend cells in small volume (200ul) of medium, remove a small amount (~50ul) of cells for freezing and plate the remaining cells?

Also, how does passage numbering work? When exactly does it increase?

What do you mean "replacing a vial of cells"?
Yup, your procedure sounds right.  You can choose to passage the cells after trypsinisation or you can freeze them down in DMSO.  Some people freeze cells in DMSO and media but we use DMSO diluted in serum (10% DMSO final concentration) and slow freeze the cells.

And your question about 'passage': https://www.google.c...iw=1366&bih=635

Our lab (*and most scientists) define 1 passage or P1 as when cells leave a monolayer culture flask/plate (via trypsinisation) and plated onto a fresh new flask - this is ONE passage.

By replacing a vial I mean restocking my supply so I don't run out of vials. I was told that I should always take some cells after trypsinisation and freeze them. So after I thaw and plate a vial (at P3) I'll grow the cells to confluency, trypsinize the plate, and freeze some of it (maybe 1/3 of the cells). This new vial will become the "replacement" for the original vial I used with the same passage number. The rest of the cells will be use for experiments.

Also, when I freeze down cells, I try to remove as much media (MEMa) as I can, but there is always 1-3 microliters keeping the pellet moist. Will that affect the cells during the freezing process?

Let me see if I understand the process of passage numbering. The passage only goes up when and only when I let cells grow to confluency and need to split them to X number of plates, right? What if I freeze down the cells instead of split and plate them? Will the vials be of the same passasge or will they be a passage higher?

Edited by Wek, 16 December 2012 - 07:08 PM.

[science noob]

Wek, on 16 December 2012 - 07:02 PM, said:

science noob, on 16 December 2012 - 06:38 PM, said:

Wek, on 16 December 2012 - 02:21 PM, said:

Could anyone give me brief explanation of the process in replacing a vial of cells when you plate it?
Do you grow the vial on a plate to ~80% confluency, trypsinize the cells, deactivate trypsin with medium, pellet the cells, resuspend cells in small volume (200ul) of medium, remove a small amount (~50ul) of cells for freezing and plate the remaining cells?

Also, how does passage numbering work? When exactly does it increase?

What do you mean "replacing a vial of cells"?
Yup, your procedure sounds right.  You can choose to passage the cells after trypsinisation or you can freeze them down in DMSO.  Some people freeze cells in DMSO and media but we use DMSO diluted in serum (10% DMSO final concentration) and slow freeze the cells.

And your question about 'passage': https://www.google.c...iw=1366&bih=635

Our lab (*and most scientists) define 1 passage or P1 as when cells leave a monolayer culture flask/plate (via trypsinisation) and plated onto a fresh new flask - this is ONE passage.

By replacing a vial I mean restocking my supply so I don't run out of vials. I was told that I should always take some cells after trypsinisation and freeze them. So after I thaw and plate a vial (at P3) I'll grow the cells to confluency, trypsinize the plate, and freeze some of it (maybe 1/3 of the cells). This new vial will become the "replacement" for the original vial I used with the same passage number. The rest of the cells will be use for experiments.

Also, when I freeze down cells, I try to remove as much media (MEMa) as I can, but there is always 1-3 microliters keeping the pellet moist. Will that affect the cells during the freezing process?

Let me see if I understand the process of passage numbering. The passage only goes up when and only when I let cells grow to confluency and need to split them to X number of plates, right? What if I freeze down the cells instead of split and plate them? Will the vials be of the same passasge or will they be a passage higher?

What I normally do is I would passage a 80-90% confluent T25 flask (25mm2) into either 3x T25 or 1xT75 or 2xT25 + few frozen vials.  If you want to be precise, you can freeze down a fixed number of cells (e.g. 1 x 10^6 per vial)

A few ul of media doesn't make a difference when you want to use the DMSO/serum freezing combination.  The crucial thing is to use DMSO.  Like I said, I use 10% DMSO in serum but I've heard people using 10% DMSO in media or even 5% DMSO.  Really depends on your cell type + how robust it is.

Different people have different ways to record passage number.  The consensus is that as long as you've trypsinised the cells from the plastic surface, the cells will be P+1.  Some people label these cells (to be frozen) as P and then label P+1 once they thaw it out and replate.  On the other hand, some would label any cells lifted from the plastic culture surface as P+1 and when you plate these down it will still remain as P+1.

[bob1]

science noob, on 16 December 2012 - 10:38 PM, said:

Different people have different ways to record passage number.  The consensus is that as long as you've trypsinised the cells from the plastic surface, the cells will be P+1.  Some people label these cells (to be frozen) as P and then label P+1 once they thaw it out and replate.  On the other hand, some would label any cells lifted from the plastic culture surface as P+1 and when you plate these down it will still remain as P+1.

I use this system - any time the cells are lifted and expanded or for freezing down add a passage #.  For instance if I took some cells at p2 and lifted them and froze them down (p+1), I would write p3 on the tube and thaw them as p3...

Also note that the different serum/DMSO/medium combinations should all work fine, though I think most people would agree that 10% DMSO and a slow freeze with a quick thaw for revival is best.  Adding DMSO straight to serum can cause precipitation of proteins in the serum, so I tend to avoid that and use 10% serum (Actually this owrks out ot be about 12% I think, with the serum that is already in the medium), 10% DMSO and 80% medium.