Lymphocyte Proliferation and BrdU - (Dec/12/2007 )
I am trying to standardise the protocol for lymphocyte proliferation using BrdU ELISA kit from Roche. I culture the PBMCs (5 X 10^4cells/well) along with mitogens in a 96 well flat bottomed plate (100ul total volume). The medium used is RPMI with 3% of heat inactivated autologous plasma. After 48hrs of incubation, I add 10uM BrdU (10ul) and incubate for another 24hrs. At the end of incubation period, I dislodge the sticking cells with a pipette and then centrifuged at 300g for 10mins (as mentioned in the product insert).
Problems - 1. This speed is not suffecient as I see a lot of cells floating in the culture supernatant after centrifugation.
2. There is lot of well to well variation.
3. The cells stick to one side of the well. This I feel reduces the accessibility of the bound BrdU to antibodies.
4. The background values are very high.
5. I dont see an increase in proliferation with mitogens (Con A - 5 and 10ug/ml, PHA - 5 and 10ug/ml).
Can any of you please guide as to what can be done to avoid all these?
Problems - 1. This speed is not suffecient as I see a lot of cells floating in the culture supernatant after centrifugation.
2. There is lot of well to well variation.
3. The cells stick to one side of the well. This I feel reduces the accessibility of the bound BrdU to antibodies.
4. The background values are very high.
5. I dont see an increase in proliferation with mitogens (Con A - 5 and 10ug/ml, PHA - 5 and 10ug/ml).
Can any of you please guide as to what can be done to avoid all these?
Q1. they may be cell debris
Q2. When seeding the cells,
1) mix the cells by inversion gently in a 50ml tubes.
2) pour the mixture into a 100mm culture dish
3) use a good mutichannel pipette to aliquot quickly
4) swirl the culture dish after seeding half the plate
5) aliquot the rest.
To minimize the variation among triplicates, try to aliquot the cell into the triplicate well at the same time.
I normally divide the plate into four columns (each contains 3x8wells),then use a multichannel pipette with 6 tips
and aliquot the cells mixture into half the row (say A1-A6, A8-A12, B1-B6, B7-B12).
Since the cells do settle a little over time in the culture dish, this method can ensure the same cell density in
each triplicates.
Q 3. please check link http://www.protocol-online.org/forums/inde...21347&st=15
Q4. how high are the background value?
Q5. the concentration of Con A and PHA are correct.
Hope this may help.