Danijela, December 10

10 Dec

Last week was the week 5 of the 6 week study, round 2. Together with Casey we analyzed CVM samples from 4 donors on M, W and F. On Thursday we also analyzed the same four CVM samples with SP. With Casey being around I got an opportunity to spend more time with IDL analysis. Shown bellow are 4 graphs that I have generated independently for a donor 004 in presence of SP over the four week course. I think that it would be good to have another more experienced IDL user to plot the data for the same user to see if we are reaching the same conclusions or someone sitting with me through one set of data to see if my reasoning is on spot. This week is the last week of the study and I will be spending much more time analyzing the data by IDL and working on First Infected Cell Project.ImageImageImageImage

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Danijela, December 10, 2012

10 Dec

Last week was the week 5 of the 6 week study, round 2. Together with Casey we analyzed CVM samples from 4 donors on M, W and F. On Thursday we also analyzed the same four CVM samples with SP. With Casey being around I got an opportunity to spend more time with IDL analysis. Shown bellow are 4 graphs that I have generated independently for a donor 004 in presence of SP over the four week course. I think that it would be good to have another more experienced IDL user to plot the data for the same user to see if we are reaching the same conclusions or someone sitting with me through one set of data to see if my reasoning is on spot. This week is the last week of the study and I will be spending much more time analyzing the data by IDL and working on First Infected Cell Project. ImageImageImageImage

Danijela November 30, 2012

30 Nov

This week was all about mucus and IDLing. As of today we are completing a week 4 of the 6 week study and it is very nice to have Casey! We no longer have issue with CVM samples and everything is running smoothly. Having Casey around to help with CVM imaging has allowed me to finally sit down and do some IDLing. I got lots of useful advice from Dan, Gianguido, Meegan and Casey and I am starting to not only get a big picture but to understand details. In discussing with Gianguido we have discovered several things that would need modification either in code or in the way that we analyze the things.. while it is true that one can rather quick start producing data by simply plugging the previously generated code much more decision making and troubleshooting needs to be done do produce more useful results. Also we found out that some of the things are taking much longer to do when working on data remotely (I am currently using Doug’s PC to access data on Wumbus) so brought up some more issues that I will be getting Gianguido’s help to sort out. I hope to be able to include some nice graphs in my blog next week.

2 Nov

expressing mcherry

293T mCherry expressing cells

2 Nov

293T mCherry expressing cells

test

Danijela October 26, 2012

26 Oct

6 week study, round 1:

This was the last week of the 6 week study, round 1. I received two CVM samples (donors 001 and 002) on M, W and F. Both samples were mixed with virus/beads mixture and imaged on Laverne. I used the following viruses: CladeC, CM235, Bal, VLP (all in mCherry) and delta envelope in GFP. Imaging went smoothly and I will be busy IDLing most of the next week so that we can obtain enough information before we proceed with the round 2 on November 5. I also imaged one CVM sample from the Core study for Shanon.

First infected cell project:

I infected p42 cells with VSVg lich and completed a time course study to assess luciferase and mCherry expression. For both, I seeded p42 cells in triplicates in 96 well plates and pulled them out at 0 hours, 1 day 2 days and 3 days post infection. Cells that will be analyzed by flow to measure mCherry expression were fixed in formaldehyde/PBS and stored at 4 degrees and cells that will be analyzed by luminometar to measure luciferase expression were lysed in lysis buffer and stored at minus 80C. In addition I also attached p42 cells infected with virus to cover slips and I fixed them at the same time points with PIPES/formaldehyde and mounted them onto the slides to be analyzed by microscopy. I will perform flow, luciferase assay and microscopy on Monday to test if luciferase and mCherry are being expressed at the same time. I wanted to thank Z and Meegan for their advise on tissue culture procedures. Beth and Erin helped complete this work.

Additionally I got trained to use META system at the Cell Imaging Facility early in the week. On Wednesday Beth and I obtained some 293T cells expressing mCherry and after we fixed them at the cover slips we took them over to do spectral imaging on our own. Looking at the cells under the microscope we saw many cells glowing red, however only for some of them we got a single peaks of fluorescence at 610 nm corresponding to mCherry. With Spectral Imaging system and program down we are signed up at the Facility again on Monday to look at our SIV lich infected p42 cells at various time points. Also, as Dan was able to find many cells glowing red in mCherry but not TRITC in cervical tissue of infected macaque we will spectral imaging on these sections to see if the cells are infect mCherry positive. I will include images and graphs from the Spectral Imaging on Monday as I transfer them from the server at the facility.

I also met with Doug to discuss cloning strategy for the luciferase:mCherry fusion protein. The plan is to use RRL GFP WPRE plasmid with CMV promoter and to excise GFP and replace it with mCherry that I will obtain from the RLICH RRL Luc IRIS Cherry plasmid. I will next amplify luciferase from the PGL 4.10 Luc2 vector and clone it in the RRL mCherry WPRE plasmid previously generated. Z will help me next week to hunt down these plasmids and we will go over the cloning strategy in more details to make sure the work is feasible.

Danijela October 5, 2012

5 Oct

This week was all centered around the First Infected Cell Project.

I sectioned tissue from female monkeys we processed two weeks ago.  Thus far I cut two different blocks of vagina, one rectum and one cervix, all which were suppose to have a very strong luminescence signal. The first vagina block did not have any stratified layer so we got worried that we may be not positioning the tissue correctly in OCT for freezing. When processing the second vagina block, rectum and cervix I made sure to check that epithelia is looking healthy before proceeding with fixing and staining. I fixed the tissue and stained it with E-cadherin (for vaginal and cervical tissue) and Ck7 (for rectal tissue) and DAPI.  In all three instances the epithelia was fine. Of note is that even though macaques are suppose to have much thicker stratified layer than human, the epithelia was quite thin due to the fact that these are only 1 year old macaques that we are dealing with. I spend some time on both Jake and Elwood making sure that I know how to use them correctly to search for the mCherry expressing cells. Starting next week with help of Erin I will scan through close to 50 slides while working on sectioning other hot blocks and staining them further. Also, Dan infected some cells with mCherry producing virus and I put some of the cells on the tissue, incubated them at 37 degrees C for an hour and proceeded with fixing and staining as previously done by Katharina and Ann. I will also analyze these slides in the coming week.

Today we received tissue from two more male macaques RKv14 and RLr14. We received recta, colons and ceca. Both which were infected with JRFL virus rectally. Macaque RKv14 had a bit of glowing recta but RLr14 recta did not seem to have much real signal. Colons for both monkeys had signal that was very focused. Ceca from the RKv14 macaque was negative as expected, however ceca from RLr14 monkey did not look like ceca at all. It was much bigger in size in comparison to 5 other ceca we saw and it very much resembled colon. Also it had very strong luminescence signal. Please see images attached (images 1 and 2 are recta from two animals, images 3 and 4 are colons from 2 animals and images 5 and 6 are what was sent labeled as ceca from two animals). Next week we are not receiving any monkeys so I will focus my efforts to sectioning and staining more tissue, searching of red cells, training Erin to help with most of these things. I will also talk in more details with Casey and Shanon about how they process mucus so that I can help process samples in the near future. I will be also spending some time preparing the article for the Virology JC for the upcoming Friday. Hope everyone has a good weekend.

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