Monday: Labor day
Tuesday 6-1-99: Met with Dr. Christensen to discuss the WIP meeting on thursday. He went through the information he will be presenting in the meeting, explaining the experiment and answering questions to me. Later I started making plate plans and protocols for the experiment he approved!
Wednesday 6-2-99: The plate plan
Thursday 6-3-99: The long awaited presentation!!! It went without incident and everyone there seemed to agree that the experiment was do-able but it would have to be done carefully so that the ICAM doesn't induce other cytokines. All the preparations finally came to use today when the rat surgery and macrophage extraction went without incident. Upon counting, looks like I lost some cells and it was confirmed when I only got 2 million cells. I had to set up 3 plates, time points: 1,3,5 days in GM-CSF incubation. This will all become clearer with the plate plans and experimental details below. REFERRED TO AS EXPERIMENT 1. Probably do another rat tomorrow and it should show that the results are reproducible and allow more time points to be tested. NOTE: The 30,000 and 50,000 cell wells have been switched on the actual plates for EXPERIMENT 1. I will switch the results.
Experimental design: The number represents the amount of cells/well. Each well will have 100uL and a final concentration of 1ng/ml of GM-CSF will be used in plates 2-8. Plate 1 will have no GM-CSF. Plates 2-8 will be read using the MTS assay on Day1 through Day7. 6 replicates were used. The results will be posted daily. Full graphic results will be posted in the appropriate week once Day 7 data is analyzed.
Plate 1
A1
No GM-CSF day 0 MTS assay
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
Plate 2 As stated
earlier, plates 2-8 are for time points day 1 through day 7.
A1
GM-CSF incubation at 1ng/ml. 100ul/well MTS assay
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
| 0 | 1000 | 5000 | 10000 | 30000 | 50000 | ||||||
Friday 6-4-99: Another rat was sacrificed for macrophages.
I was able to do six plates this time. The rest of the macrophages
were given to another researcher. The plates were set up as earlier
but the time points I will use are 1,3,4,5, and 7 days. THIS EXPERIMENT
WILL BE LABELED EXPERIMENT 2. The plating proceeding without incident.
I read up on the GM-CSF we were using and although mouse GM-CSF does act
across species lines, it seems that it is not stable when freeze/thawed.
Therefore, I will have to make smaller aliquots of GM-CSF next time I freeze
some. Right now, there is 20uL per aliquot but 10 should be sufficient.
Also did the MTS
assay today for the day 1 time point for mac proliferation of experiment
1 above.
RESULTS: Day 1 time point showed that the MTS assay is not very
sensitive for cells at low concentration but at 30,000 and 50,000 cells
per well worked, showing a consistantly higher absobance after 60 minutes
of MTS/PMS incubation. 60 and 80 mins seem to be the best times.
Dr. Christensen has been nice enough to come in on the weekend to do the
time points since I live over 1 hour away. Wrote up a MTS assay protocol
for him. This was a REALLY long day.
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