PMA/ionomycine activated cells

Hello there,

I have a problem.
I have frozen PBMCs, cultured for 24 hours, activated with PMA 25 ng/mL and ionomycine 1 µg/mL for 4 hours, in the presence of brefeldin-A to inhibit the secretion of the synthesized cytokines.
After 4 hours, the cells are harvested. I see a nice pellet.
some surface markers are labeled for flow cytometry, cells are washed, centrifuged. I still have a nice pellet.
I fix with PFA 1% 15 minutes at room temperature. centrifuge. Still have a pellet.
permeabilize with saponin 0.2% (in PBS plus 1% BSA, 5 mM EDTA, 0.02% Na azide) in the presence of the antibodies, for 1 hour at 4°C. after centrifugation : no more pellet.
However, the cells that were not activated by PMA ionomycin are still here.

what did i do wrong?
Should I optimize the saponin concentration with the activated cells?
is it right to let ionomycin for 4 hours, or should i wash earlier and harvest the cells after 4 hours?

thanks in advance for your advices

Missele

QUOTE (Missele @ Jan 15 2007, 04:15 PM)

Hello there,

I have a problem.
I have frozen PBMCs, cultured for 24 hours, activated with PMA 25 ng/mL and ionomycine 1 µg/mL for 4 hours, in the presence of brefeldin-A to inhibit the secretion of the synthesized cytokines.
After 4 hours, the cells are harvested. I see a nice pellet.
some surface markers are labeled for flow cytometry, cells are washed, centrifuged. I still have a nice pellet.
I fix with PFA 1% 15 minutes at room temperature. centrifuge. Still have a pellet.
permeabilize with saponin 0.2% (in PBS plus 1% BSA, 5 mM EDTA, 0.02% Na azide) in the presence of the antibodies, for 1 hour at 4°C. after centrifugation : no more pellet.
However, the cells that were not activated by PMA ionomycin are still here.

what did i do wrong?
Should I optimize the saponin concentration with the activated cells?
is it right to let ionomycin for 4 hours, or should i wash earlier and harvest the cells after 4 hours?

thanks in advance for your advices

Missele

PMA and ionomycin in parallel applied and in these conc for 24h is harsh to cells; these agents trigger very much in the cell and there are, at least, strong rearrangements of the cytoskeleton; this profoundly destabilizes cells, saponin seems give them the rest; may be many of them are pro-apoptotic/pro-necrotic or even apoptotic/necrotic; one should check this...

QUOTE (The Bearer @ Jan 15 2007, 08:15 PM)

QUOTE (Missele @ Jan 15 2007, 04:15 PM)

Hello there,

I have a problem.
I have frozen PBMCs, cultured for 24 hours, activated with PMA 25 ng/mL and ionomycine 1 µg/mL for 4 hours, in the presence of brefeldin-A to inhibit the secretion of the synthesized cytokines.
After 4 hours, the cells are harvested. I see a nice pellet.
some surface markers are labeled for flow cytometry, cells are washed, centrifuged. I still have a nice pellet.
I fix with PFA 1% 15 minutes at room temperature. centrifuge. Still have a pellet.
permeabilize with saponin 0.2% (in PBS plus 1% BSA, 5 mM EDTA, 0.02% Na azide) in the presence of the antibodies, for 1 hour at 4°C. after centrifugation : no more pellet.
However, the cells that were not activated by PMA ionomycin are still here.

what did i do wrong?
Should I optimize the saponin concentration with the activated cells?
is it right to let ionomycin for 4 hours, or should i wash earlier and harvest the cells after 4 hours?

thanks in advance for your advices

Missele

PMA and ionomycin in parallel applied and in these conc for 24h is harsh to cells; these agents trigger very much in the cell and there are, at least, strong rearrangements of the cytoskeleton; this profoundly destabilizes cells, saponin seems give them the rest; may be many of them are pro-apoptotic/pro-necrotic or even apoptotic/necrotic; one should check this...

Thanks for your answer the bearer,
however, I incubated with PMA and iono for only 4 hours. I saw in the litterature some activations of 4 to 6 hours in the same conditions.
However, there are maybe some tricks with the medium in which you should incubate, or maybe wash ?

QUOTE (Missele @ Jan 16 2007, 10:05 AM)

QUOTE (The Bearer @ Jan 15 2007, 08:15 PM)

QUOTE (Missele @ Jan 15 2007, 04:15 PM)

Hello there,

I have a problem.
I have frozen PBMCs, cultured for 24 hours, activated with PMA 25 ng/mL and ionomycine 1 µg/mL for 4 hours, in the presence of brefeldin-A to inhibit the secretion of the synthesized cytokines.
After 4 hours, the cells are harvested. I see a nice pellet.
some surface markers are labeled for flow cytometry, cells are washed, centrifuged. I still have a nice pellet.
I fix with PFA 1% 15 minutes at room temperature. centrifuge. Still have a pellet.
permeabilize with saponin 0.2% (in PBS plus 1% BSA, 5 mM EDTA, 0.02% Na azide) in the presence of the antibodies, for 1 hour at 4°C. after centrifugation : no more pellet.
However, the cells that were not activated by PMA ionomycin are still here.

what did i do wrong?
Should I optimize the saponin concentration with the activated cells?
is it right to let ionomycin for 4 hours, or should i wash earlier and harvest the cells after 4 hours?

thanks in advance for your advices

Missele

PMA and ionomycin in parallel applied and in these conc for 24h is harsh to cells; these agents trigger very much in the cell and there are, at least, strong rearrangements of the cytoskeleton; this profoundly destabilizes cells, saponin seems give them the rest; may be many of them are pro-apoptotic/pro-necrotic or even apoptotic/necrotic; one should check this...

Thanks for your answer the bearer,
however, I incubated with PMA and iono for only 4 hours. I saw in the litterature some activations of 4 to 6 hours in the same conditions.
However, there are maybe some tricks with the medium in which you should incubate, or maybe wash ?

ok, my fault, sorry, 24h were the preculturing of the cells;
PMA is a very strong modulator in cells; in combination with ionomycin it may become cytotoxic; so, if I would have to do these experiments, I would try several concentrations of PMA and ionomycin; PMA starts doing effects even at pmolar range; your ionomycin conc is okay and often documented but incubation for several hours burdens a cells (free calcium normally oscillates after stimulation of a cell, but ionomycin supports permanent high levels of Ca2+);
I would let ionomycin unchanged for short-term incubation and lower the PMA conc; short-term effects are rapid concerning phosphorylation events and take place in a range between 0 to 20-30 min; so may be it would do for the first experiments to analyze short-term effetcs;

for long-term incubation I would also lower ionomycin conc and try to use lower PMA conc

PMA/ionomycine activated cells - intracellular staining after saponin permeabilisation (Jan/15/2007 )