Immunofluorescense of floating cells - How to IF the floating cells during apoptosis? (Aug/28/2008 )
hi,
When I induce apoptosis in my cells they start to float and are not adherent anymore. they usually get washed away during the washing steps. what should I do?
can I keep them attached to the coverslips some how?
I'm just stuck 
-Curtis-
I'd be careful about trying to do IF on the floating cells. These are completely dead (or really close) and some people will be skeptical of data coming from this population. Rather, I would try to be as gentle as possible. When I work with cells arrested with nocodazole, they are barely adherent and come off easily. These I only wash once and am careful to let the pbs go down the side of the well and not directly on the cells. I do my best to not bump anything and if you can, fixation in paraformaldehyde seems to maintain more cells on the glass than methanol.
Are you using the best coverglass for these cells (ie: collagen coated or poly-L coated)? Can you induce for a shorter time? This may allow more cells to remain adherent but you can still see an effect. Otherwise, if you must use the floating cells then treat this like a suspension cell culture. There are tons of protocols available for doing IF on cells in suspension. You essentially need to spin the cells down onto a coverslip and immediately fix. Pretty easy and I've done something similar with egg extracts.
-rkay447-
QUOTE (rkay447 @ Aug 28 2008, 06:17 AM)
I'd be careful about trying to do IF on the floating cells. These are completely dead (or really close) and some people will be skeptical of data coming from this population. Rather, I would try to be as gentle as possible. When I work with cells arrested with nocodazole, they are barely adherent and come off easily. These I only wash once and am careful to let the pbs go down the side of the well and not directly on the cells. I do my best to not bump anything and if you can, fixation in paraformaldehyde seems to maintain more cells on the glass than methanol.
Are you using the best coverglass for these cells (ie: collagen coated or poly-L coated)? Can you induce for a shorter time? This may allow more cells to remain adherent but you can still see an effect. Otherwise, if you must use the floating cells then treat this like a suspension cell culture. There are tons of protocols available for doing IF on cells in suspension. You essentially need to spin the cells down onto a coverslip and immediately fix. Pretty easy and I've done something similar with egg extracts.
Are you using the best coverglass for these cells (ie: collagen coated or poly-L coated)? Can you induce for a shorter time? This may allow more cells to remain adherent but you can still see an effect. Otherwise, if you must use the floating cells then treat this like a suspension cell culture. There are tons of protocols available for doing IF on cells in suspension. You essentially need to spin the cells down onto a coverslip and immediately fix. Pretty easy and I've done something similar with egg extracts.
as always I thank you
-Curtis-
QUOTE (rkay447 @ Aug 28 2008, 07:17 AM)
I'd be careful about trying to do IF on the floating cells. These are completely dead (or really close) and some people will be skeptical of data coming from this population. Rather, I would try to be as gentle as possible. When I work with cells arrested with nocodazole, they are barely adherent and come off easily. These I only wash once and am careful to let the pbs go down the side of the well and not directly on the cells. I do my best to not bump anything and if you can, fixation in paraformaldehyde seems to maintain more cells on the glass than methanol.
Are you using the best coverglass for these cells (ie: collagen coated or poly-L coated)? Can you induce for a shorter time? This may allow more cells to remain adherent but you can still see an effect. Otherwise, if you must use the floating cells then treat this like a suspension cell culture. There are tons of protocols available for doing IF on cells in suspension. You essentially need to spin the cells down onto a coverslip and immediately fix. Pretty easy and I've done something similar with egg extracts.
Are you using the best coverglass for these cells (ie: collagen coated or poly-L coated)? Can you induce for a shorter time? This may allow more cells to remain adherent but you can still see an effect. Otherwise, if you must use the floating cells then treat this like a suspension cell culture. There are tons of protocols available for doing IF on cells in suspension. You essentially need to spin the cells down onto a coverslip and immediately fix. Pretty easy and I've done something similar with egg extracts.
rkay, I forgot to ask,....I've never used Poly L Lysine although i read it everywhere and also my friends told me to, but I honestly have not....I just saw that HeLa cells grow much faster on the glass coverslip than on the bottom of the flask, 6-well plate or petri dish,.....I can clearly see that and that is the main reason why I never used it......the other reason is that I don't have Poly L Lysine
.....so if it is that necessary I will order and buy it ! what do you say?.........I really don't want the apoptotic cells to get washed off.....and also I am already aware that I should do the experiments just before the blebbing happens (as you mentioned in your post)the other thing is that, I made my mounting buffer myself by adding PBS/Glycerol at a ratio of 1:9....but I can see a lot of bubbles under the slide after mounting...how can I get rid of those?.....just by pressing the coverslip?....I'm asking this because once I did mounting with Invitrogen's ProLonging agent and after 24 hours I got really sharp images....but I don't have much money to buy it, although it might be very cheap for you....I just want to know how you do the mounting?
-Curtis-
QUOTE (Curtis @ Aug 28 2008, 04:09 PM)
QUOTE (rkay447 @ Aug 28 2008, 07:17 AM)
I'd be careful about trying to do IF on the floating cells. These are completely dead (or really close) and some people will be skeptical of data coming from this population. Rather, I would try to be as gentle as possible. When I work with cells arrested with nocodazole, they are barely adherent and come off easily. These I only wash once and am careful to let the pbs go down the side of the well and not directly on the cells. I do my best to not bump anything and if you can, fixation in paraformaldehyde seems to maintain more cells on the glass than methanol.
Are you using the best coverglass for these cells (ie: collagen coated or poly-L coated)? Can you induce for a shorter time? This may allow more cells to remain adherent but you can still see an effect. Otherwise, if you must use the floating cells then treat this like a suspension cell culture. There are tons of protocols available for doing IF on cells in suspension. You essentially need to spin the cells down onto a coverslip and immediately fix. Pretty easy and I've done something similar with egg extracts.
Are you using the best coverglass for these cells (ie: collagen coated or poly-L coated)? Can you induce for a shorter time? This may allow more cells to remain adherent but you can still see an effect. Otherwise, if you must use the floating cells then treat this like a suspension cell culture. There are tons of protocols available for doing IF on cells in suspension. You essentially need to spin the cells down onto a coverslip and immediately fix. Pretty easy and I've done something similar with egg extracts.
rkay, I forgot to ask,....I've never used Poly L Lysine although i read it everywhere and also my friends told me to, but I honestly have not....I just saw that HeLa cells grow much faster on the glass coverslip than on the bottom of the flask, 6-well plate or petri dish,.....I can clearly see that and that is the main reason why I never used it......the other reason is that I don't have Poly L Lysine
.....so if it is that necessary I will order and buy it ! what do you say?.........I really don't want the apoptotic cells to get washed off.....and also I am already aware that I should do the experiments just before the blebbing happens (as you mentioned in your post)the other thing is that, I made my mounting buffer myself by adding PBS/Glycerol at a ratio of 1:9....but I can see a lot of bubbles under the slide after mounting...how can I get rid of those?.....just by pressing the coverslip?....I'm asking this because once I did mounting with Invitrogen's ProLonging agent and after 24 hours I got really sharp images....but I don't have much money to buy it, although it might be very cheap for you....I just want to know how you do the mounting?
Here we use fingernail polish to fix the coverslip on the slide, but sealing the coverslip completely, thus making it airtight allows formation of those bubbles. I rather store my slides in the fridge.
-raghar-