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Which cell line produce cytokine? - (Mar/06/2009 )

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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

-klinmed-

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?

-acute-

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 g/ml). Store aliquots at -20 oC. Use 0.05 ml in 5 ml culture.

-klinmed-

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 g/ml). Store aliquots at -20 oC. Use 0.05 ml in 5 ml culture.



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?

-acute-
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