Version of file for indexing 8-22-12

2015-01-13

azim58 - Version of file for indexing 8-22-12

Summary of Ova CTL Experiment 8-21-12
A CTL chromium release assay with B16F10 target cells pulsed with
SIINFEKL peptide from the
Ova antigen and effector cells from Ova immunized mice did demonstrate
that the target cells were
killed in a dose dependent manner with specific p values compared to
controls. However, the current
CTL chromium release assay protocol needs to be optimized. This is
actually the 2nd time I performed
the chromium release assay. The first time, I made a pipetting error
which affected many samples, and I
also observed some results I could not fully explain. I suspected but
could not prove that DMSO alone
was killing my target cells. In this experiment, I had all of the
controls necessary to prove that this was
the case. There are still some things I wasn’t fully expecting from
this experiment though. I did not
expect that effector cells would kill non-peptide pulsed tumor cells in a
dose dependent manner. Now
that I see the result this makes sense to me though since effector cells
probably have some general
ability to recognize some features of tumor cells. I also did not expect
the negative control peptide
sample to exhibit greater killing at low E:T ratios and less killing at
higher E:T ratios.
Note that in this protocol my target cells were incubated with the
chromium for about 5 hr
rather than the 1-2 hr that the protocol suggests. This happened because
I thought I had enough
effector cells from the previous experiment, but this turned out not to
be the case. Therefore, I
obtained some more splenocytes from another Ova immunized mouse which
took time.
I have two sets of data one for a 6 hr incubation plate and one for an
overnight plate. Note that
all p values were calculated by comparing a certain E:T sample with the
corresponding target with
peptide dissolved in water and DMSO or DMSO only.
E:T Ratios
E:T
Ratio
Target
Cell
Number
Effector
Cell
Number
100 1.00E+04 1.00E+06
30 1.00E+04 3.00E+05
10 1.00E+04 1.00E+05
3 1.00E+04 3.00E+04
Sample Name Key
TONPND = Target cells only not pulsed no DMSO
TONP= Target cells only not pulsed
TOP = Target cells only pulsed
TONCP = Target only negative control pulsed (negative control = CPV172
peptide)
EO = Effector Cells only
EOP = Effector cells only with peptide
EONCP = Effector cells only with negative control peptide
TNPEX = Targets non-pulsed with effectors at E:T ratio X:1
TNCPEX = Targets negative control pulsed with effectors at E:T ratio X:1
TPEX = Targets pulsed with effectors at E:T ratio 100:1
6 hr Incubation Results
Overnight Incubation Results
Ways to Optimize Protocol
· Use as little DMSO as possible or even no DMSO if the peptide can be
soluble without it.
Alternatively, try incubating the target cells with peptide in a larger
volume (rather than
approximately 100 uL).
· Resuspend all pelleted target cell samples in a specific volume. The
protocol I followed said to
take away the supernatant and leave about 100 uL left. However, I think I
ended up with
varying volumes for my 3 types of target cells (non-pulsed, ova peptide
pulsed, and NC peptide
pulsed). I think it would have been better to take away just about all of
the liquid and
resuspend all target cells in a known volume (probably 1-5 mL RPMI rather
than 100 uL). The
media was starting to change colors after the incubations, and I think
the cells may not have had
enough nutrients in the small 100 uL volume of media.
· Don’t let the target cells incubate with chromium for longer than
1-2 hr.
· When transferring 100 uL of supernatant to the Luma plate for
counting, make sure the tips are
firmly attached and a full 100 uL is transferred to the Luma plate for
all of the tips on the
multichannel.

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