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Monday  5-10:  Interview with Dr. Christensen about the research position.  Looks like I have the job.  The research should be difficult but interesting.

Wednesday 5-12:  Got several papers of background reading.  Called Sigma.  Apparently the ANTI-GM-CSF antibody we need to proceed with experiment one is not yet available even though we back ordered it over two monts ago.  Expected date of arrival is June.

Thursday 5-13:   Obtained backgroud reading for second experiment dealing with soluble ICAM.  This experiment looks much longer.  Did backgroud reading for preliminary experiments we (Donna and I) are starting tomorrow.  Set up plate etc for tomorrow's MTS assay and also starting GM-CSF proliferation assay (proliferation also measured using MTS).  Found a rat we could use for macrophages. Made standard solutions of PBS for tomorrow.

Friday 5-14:  Well, the first thing I learnt today is that rats are a lot bigger than the mice I worked with last year.  This rat was older than most--and was huge!!  The rat had to be caught in a plastic bag because nobody could hold such a large rat.  Click here for pentabarbitol killing procedures. Usually 1cc. of pentabarbitol is enough but after 1 cc, we had to administer .3 cc more.  Click here for Bronchio-Alveolar Lavage Procedures that were performed to extract macrophages. The Macs. were extracted using a 10cc seringe.  Approx. yield was 6 million cells.  Macs were isolated from some red blood cells that were present by washing and resuspending the cells in PBS twice. This works because macs adhere to plastic.   These cells were plated in a 96 well plate as follows:
note: there is a total of 100 ul/ of media well.
 

96 well plate of 5/14 experiment for MTS assay using Macs. (number of cells/well)

0	100	500	1E3	5E3	1E4	5E4	1E5	5E5
0	100	500	1E3	5E3	1E4	5E4	1E5	5E5
0	100	500	1E3	5E3	1E4	5E4	1E5	5E5

This plate was incubated in 7% CO2, 37C for one hour.  The MTS/PMS solutions must be thawed. The ratio of MTS/PMS needed is 2ml/100ul.  20 ul of this goes into every well.  The plate was covered with alum. foil and incubated again.  I checked every 15-20 mins for visual color differences to appear.  At 60 mins, I took the first reading using the ELISA plate reader set at 490 nm.  The results obtained (absorbance vs. number of cells) was approximately linear.  However more points are needed at higher cell numbers in the plate to make sure that the assay is linear at high cell numbers.

We also set up a second experiment which involved the proliferation of Macrophages based on the amount of GM-CSF present in each well.  The numbers correspond to ng/ul of GM-CSF present.  The 96 well plate looked like this:
 

Mac. proliferation based on amount of GMCSF present  using MTS assay

0	0	0	0	0	0	0	0	0	0	0	0
.3	.3	.3	.3	.3	.3	.3	.3	.3	.3	.3	.3
1	1	1	1	1	1	1	1	1	1	1	1
3	3	3	3	3	3	3	3	3	3	3	3
10	10	10	10	10	10	10	10	10	10	10	10

 
The stock GM-CSF solution is 5ng/ml.

We incubated this over the weekend.  Please see next week's information for results of this experiment.