I need some help regarding bioassay of G-CSF using MNFS-60 cell line. My protocol is as follows:
1) MNFS-60 cell line is maintained in 10% RPMI 1640 with 0.05mM 2-Mercaptoethanol. ( Note: To get this 0.05mM 2-Mercaptoethanol when I open 500ml bottle of RPMI 1640 that time only I add 1.7µl of 2-Mercaptoethanol in it).
2) I subculture the cell line in the same media mentioned above and incubate for 48 hrs in CO2 incubator.
3) For Bioassay I prepare Assay media (i.e. 10% RPMI 1640 without 2-Mercaptoethanol).
4) Then dilute G-CSF ranging from 800 IU/ml to 0.781 IU/ml in assay media.
5) After preparation of sample, add 50µl of assay media and 50µl spls in triplicates in 96well plate.
6) Also in the mean time centrifuge the cell at 125g for 10mins then decant supernatant and resuspend pellet in 4ml of assay media & again centrifuge.
7) Then prepare the cell suspension of 7X105cell/ml and add 2-Mercaptoethanol to a final conc. of 0.1mM
8) Add 50µl of the prepared cell suspension to each well and incubate the plate for 48 hrs in CO2 incubator.
9) After incubation add 20µl of CCK-8 Kit and again incubate for 4 hrs and take absorbance at 490nm.
10) Determine the EC50 using graph pad prism software.
Now the problem I am facing while doing this assay is:
1) Sometimes I don’t get proper gradation as per dose range.
2) Sometimes I get gradation but it is completely opposite i.e. higher conc. gives lower reading n lower gives higher reading.
3) Sometimes I get proper gradation as per dose range but I don’t get required EC50 and specific activity (1 X 108 IU/mg).
I would really appreciate any kind of help.