13 replies to this topic

[acute]

Dear Friends,
I isolate PBMCs from leukemic children for apoptosis tests. As I found out I need cytokines in order to maintain them (such as IL 2 3 and 6, SCF...). But cytokines are too costy and I cannot afford it.
Then I found out that some cell lines such as 5637 (bladder carcinoma) produce some cytokines and can be used for leukemic cells proliferations. Bad luck! this cell line is not available in my country!!!!!
Have you any idea which cell lines can produce cytokines so I can use them? does the medium of HL60 (lymphoblasts) or PC12 contain some quantities of cytokines?
need so much help...thanks for any idea in advance
regrads

[CKtong]

acute, on Mar 7 2009, 12:18 AM, said:

Dear Friends,
I isolate PBMCs from leukemic children for apoptosis tests. As I found out I need cytokines in order to maintain them (such as IL 2 3 and 6, SCF...). But cytokines are too costy and I cannot afford it.
Then I found out that some cell lines such as 5637 (bladder carcinoma) produce some cytokines and can be used for leukemic cells proliferations. Bad luck! this cell line is not available in my country!!!!!
Have you any idea which cell lines can produce cytokines so I can use them? does the medium of HL60 (lymphoblasts) or PC12 contain some quantities of cytokines?
need so much help...thanks for any idea in advance
regrads

u may also try jurkat cell line(as that is a Tcell leukemic cell line). It may be gave u the cytokine u needed.
Meanwhile, i will suggest u to use cd3+cd28 microbeads(invitrogen) to expand ur cell. These bead can be reuse(though is not recommended). but need to do more works as u have to detach the beads from the culture.\
Cheers

[acute]

Thanks alot
what about coculture with a fibroblast like pc12?
I heard it can also be useful to make the cells proliferate,any idea?
thanks

[CKtong]

CKtong, on Mar 7 2009, 12:12 PM, said:

acute, on Mar 7 2009, 12:18 AM, said:

Dear Friends,
I isolate PBMCs from leukemic children for apoptosis tests. As I found out I need cytokines in order to maintain them (such as IL 2 3 and 6, SCF...). But cytokines are too costy and I cannot afford it.
Then I found out that some cell lines such as 5637 (bladder carcinoma) produce some cytokines and can be used for leukemic cells proliferations. Bad luck! this cell line is not available in my country!!!!!
Have you any idea which cell lines can produce cytokines so I can use them? does the medium of HL60 (lymphoblasts) or PC12 contain some quantities of cytokines?
need so much help...thanks for any idea in advance
regrads

u may also try jurkat cell line(as that is a Tcell leukemic cell line). It may be gave u the cytokine u needed.
Meanwhile, i will suggest u to use cd3+cd28 microbeads(invitrogen) to expand ur cell. These bead can be reuse(though is not recommended). but need to do more works as u have to detach the beads from the culture.\
Cheers

You may also use IMDM(my lab mate use that, quite good)
as for the PC12 I not sure about it..
Back to your main question....'isolate PBMCs from leukemic children for apoptosis tests.'
if that is ur purpose, I will suggest u not to induce any other mean of treatment.( not even any cytokine/ cd3?cd28 beads. sorry, I overlooked)
As you know, these treatment will affected your cells viabiliby and some may drive them into apoptosis. Is there any reason u do not use the primary source(why need to culture??)

[acute]

dear CKtong
thanks for ur reply. I want to see the drug effects on cells from various patients therefore I cannot use the primary source like HL60 or sth like this.
But without cytokines (as I see right now) my cells will lead to necrosis after a short period (one week).
I dont know what to do to maintian them more
regards

[klinmed]

acute, on Mar 6 2009, 05:18 PM, said:

Dear Friends,
I isolate PBMCs from leukemic children for apoptosis tests. As I found out I need cytokines in order to maintain them (such as IL 2 3 and 6, SCF...). But cytokines are too costy and I cannot afford it.
Then I found out that some cell lines such as 5637 (bladder carcinoma) produce some cytokines and can be used for leukemic cells proliferations. Bad luck! this cell line is not available in my country!!!!!
Have you any idea which cell lines can produce cytokines so I can use them? does the medium of HL60 (lymphoblasts) or PC12 contain some quantities of cytokines?
need so much help...thanks for any idea in advance
regrads

In the old days (before the CSFs and interleukins were cloned) my lab used phytohemagglutinin-activated PBMC conditioned medium as a source of hematopoietic growth factors. This worked well to support colony formation in semi-solid agar cultures and in leukemic blast proliferation assays.
To make the CM, culture 1E6/ml PBMC in RPMI 1640 containing 10% FCS, 1E-4M 2-mercaptoethanol and 25 µg/ml PHA-P (Sigma). Incubate at 37 oC for 7 days. Then remove cells by centrifugation, 0.22 µm filter and store -20 oC.

You need to titrate the CM to find the optimal amount for stimulation of leukemic cell proliferation ( usually around 1% CM).

See Cebon J et al. 1990 J Biol Chem 265: 4483-4491.

Hope this helps

[acute]

Dear klinmed,
thanks so much for your help. In order to try it I have some questions. Should I use CM with RPMI + 20% FBS? Can I use HL60 cells, put PHA-P and 2-mercaptoethanol on it to produce hematopoietic growth factors? Then what if the cells die in 7 days incubation (HL60 cells are growing too fast)?
Another question. I heard LPS can induce the production of inteleukins, can it be useful?
thanks again
regards

[klinmed]

acute, on Mar 15 2009, 10:48 AM, said:

Dear klinmed,
thanks so much for your help. In order to try it I have some questions. Should I use CM with RPMI + 20% FBS? Can I use HL60 cells, put PHA-P and 2-mercaptoethanol on it to produce hematopoietic growth factors? Then what if the cells die in 7 days incubation (HL60 cells are growing too fast)?
Another question. I heard LPS can induce the production of inteleukins, can it be useful?
thanks again
regards

Hi.

RPMI containing from 5 - 20 % FBS should be fine.
The PHA-P acts as a polyclonal stimulator of T-lymphocytes (present in the PBMC) which then release hematopoietic growth factors like GM-CSF, IL-4, IL-5....etc.. The 2-ME seems to help with the cultivation of lymphocytes in vitro.
HL60 is a promyelocytic leukemia line which, to the best of my knowledge, does not produce hematopoietic growth factors no matter how you treat it. In my opinion you should forget about using HL-60 conditioned medium.

[swanny]

Imho, the best database of cytokines:

http://www.copewithc...nes.de/cope.cgi

[acute]

Dear Klinmed,
thanks so much seems to work well. Could I use PHA M form instead of P too? if yes, how much should I use, I found 1:100 in papers. is it ok?
thanks

[klinmed]

acute, on Apr 15 2009, 03:58 PM, said:

Dear Klinmed,
thanks so much seems to work well. Could I use PHA M form instead of P too? if yes, how much should I use, I found 1:100 in papers. is it ok?
thanks

Good to hear that things are working.
You can use PHA-M at 10 ug/ml. Check with the manufacturers literature for optimal concentrations.
"1:100" PHA comes from the days when PHA preparations were very crude. These are not used today.

Hope this helps

[acute]

klinmed, on Apr 15 2009, 10:35 PM, said:

acute, on Apr 15 2009, 03:58 PM, said:

Dear Klinmed,
thanks so much seems to work well. Could I use PHA M form instead of P too? if yes, how much should I use, I found 1:100 in papers. is it ok?
thanks

Good to hear that things are working.
You can use PHA-M at 10 ug/ml. Check with the manufacturers literature for optimal concentrations.
"1:100" PHA comes from the days when PHA preparations were very crude. These are not used today.

Hope this helps

Thanks again
But the problem is that the ug of my PHA-M (from gibco) is unknown. I searched the invitrogen website and didnot found anything. it is just PHA-M in the size of 10 ml.
think that I should use 1:100,right?

[klinmed]

acute, on Apr 15 2009, 09:19 PM, said:

klinmed, on Apr 15 2009, 10:35 PM, said:

acute, on Apr 15 2009, 03:58 PM, said:

Dear Klinmed,
thanks so much seems to work well. Could I use PHA M form instead of P too? if yes, how much should I use, I found 1:100 in papers. is it ok?
thanks

Good to hear that things are working.
You can use PHA-M at 10 ug/ml. Check with the manufacturers literature for optimal concentrations.
"1:100" PHA comes from the days when PHA preparations were very crude. These are not used today.

Hope this helps

Thanks again
But the problem is that the ug of my PHA-M (from gibco) is unknown. I searched the invitrogen website and didnot found anything. it is just PHA-M in the size of 10 ml.
think that I should use 1:100,right?

I have not used Gibco PHA for many years. Found this protocol in the black hole of my filing system:

Phytohemagglutinin (PHA-M): (Gibco #10576-015) lyophilized, Rehydrate vial with 10 ml water to make stock (100 µg/ml). Store aliquots at -20 oC. Use 0.05 ml in 5 ml culture.

[acute]

I have not used Gibco PHA for many years. Found this protocol in the black hole of my filing system:

Phytohemagglutinin (PHA-M): (Gibco #10576-015) lyophilized, Rehydrate vial with 10 ml water to make stock (100 µg/ml). Store aliquots at -20 oC. Use 0.05 ml in 5 ml culture.
[/quote]

oooops seems that I used the PHA M in twice more concentration!
I added the CM before I store t at -20 and It worked well, But after thawing I used it again and it contained some pieces in it, I didnot understand what tthey are....can I store it at -4?
is 1% CM enough? or shoud test the high percentages too?(like 20 or 10%)
another question...when I add the 2ME and CM to my blasts, they are going to become very smal...each one seems to divide into 2to 4 spheres that seems to be cells but their size are tooo small, could you explain me what they are?