Monday, January 27, 2014

Adventures in Lentivirusland

I've already written a bit about my troubles regarding the packaging of my lentiviral particles. At the end of my last lentiviral post I had figured out that the packaging of my CherryPicker vector was delayed and optimal collecting time was around 60 hours and not 48 hours. Additionally, I found that the amount of virus-associated p24 (lentivirus capsid) was highest in my target cell medium making harvesting and subsequent transduction quite easy. The problem was, however, that the amount of virus-associated p24 capsid only tells you how much virus you have in your supernatant. It does not indicate the amount of infectious virus in your sample and that's pretty much what you care about when you're harvesting lentiviral supernatant. 

Before, when I was using GFP as a reporter gene I would estimate the lentiviral titers by flow cytometry (FACS). I would transduce Huh7 cells in a serial dilution and then analyze the percentage of green cells in a FACS machine and then calculate transduction units per milliliter. Since I don't currently have access to a FACS machine to detect mCherry I thought that I could maybe make use of the TCID50 method to estimate the titer.


I diluted my newly collected lentiviral supernatant into a 96 well plate (similar as shown in the picture) and evaluated what wells still had fluorescent mCherry signal. Then I estimated the titer using the Spearman & Kaerber algorithm. I also estimated the titer of some GFP viruses I had along with my previous CherryPicker stocks so I had something to compare to. 

Not only does this method estimate the titer of my lentiviral stocks to be quite similar to what I had already estimated with either p24 ELISA and/or FACS but it also showed that the CherryPicker viruses that I collected after 60 hours had a very high titer. Higher than all of the previous CherryPicker stocks and even the GFP stock. 

So it seems that I have solved my lentiviral crisis. At least, this lentiviral crisis. I currently have some other ones I might share later. 

Soon I will also post a little bit about my musical guilty pleasure. 

Until next time!
Hüldi

Monday, January 13, 2014

A wonderful assortment of Christmas cookies


Dear friends

Quite some time has passed since my last post and thus, with delay, I wish you all a very happy and wonderful New Year - and I hope you will keep following our blog with curiosity and interest. I was pretty busy just before Christmas, as I not only had to finish important experiments in the lab and last sentences for my review; but we also had to move our lab and the flat to a new city. I was very touched by the great and emotional moments during our goodbye party - I certainly will remember all these funny chats while pipetting or splitting cells, the BBQs on the terrace with its amazing view over the city and I'm very grateful for all the experience and knowledge I acquired while working with so many different people - thanks for that!

After moving my belongings to the new flat and having unpacked all those boxes, just one week was left till Christmas and I was enjoying the time baking Christmas cookies for my family and friends. Christmas cookies have a long tradition in our family, and every year they are prepared in the same fashion concerning ingredients and shape and with the same accurateness that i learned from my mother.
This year it was my responsibility to prepare the cookies, as everyone was still working and as busy as one is around that time of the year! I have to admit, it was a lot of work to bake ten different types of cookies all by myself - but i really enjoyed the time to relax after busy weeks in the lab.

A resume of my homemade cookies:
Now, to the essential part of the blog: my different type of cookies - i will shortly describe each of them, so you can get inspired for next Christmas and if so, just contact me and i will be happy to share recipes and experience with you;-). One of my favorite cookie is the "spetzbueb", which is made of a very thin dough mainly containing butter, eggs and flour and whatever jam you like. I prefer, as the original recipe suggests, Raspberry Jam. If you manage to cut the cookies as thin as possible, they will melt in your mouth;-)

In the upper right corner of the plate you find a cookie based on egg white (like meringue), dates and almonds. This is the first time I ever made them and they were the favorites of many people - the foamy egg white makes them rather light for a cookie and the pieces of dates give them a very exquisite taste. Just bake them until the are slightly colored - otherwise they become dry and crumbly very fast. As many people became fond of them I will recycle the recipe also during the year for birthdays or other events, I am thinking to prepare an alternative with dried apricots or with figs - how does that sound to you?

On any Christmas-Cookie plate, cinnamon-stars should always be present. If you don't bake them too long they are still nicely humid and the taste of cinnamon will inspire you and get you in a very "Christmassy" mood.  You can also modify the icing by adding cinnamon which will intensify the flavor of the cookie a lot. I advise to not cut the stars to small, as they not only become dry much faster, but you also need much more time to ice them;-)

In the lower right corner you will see dried apricots coated with dark swiss chocolate. They are very easy and fast to prepare - but nevertheless taste lovely! They always remind me of that party night where we prepared a chocolate fondue and dipped all kinds of fruits into dark chocolate - for me just the perfect combination.

The brown cookies called "Basler Brunsli" are said to originally come from Basel and are a combination of dark chocolate and almonds, with a drop of cherry liquor. You like them or you don't - either way, they will be present on my Cookies plate every year - you can't meet the taste of everyone i guess;)

In line with the pistachio cake i posted some months ago, I intended to implement this idea into a Cookie and was happy to see that in its newest issue, Betty Bossy had the same idea and she added small pieces of orange peel ;-). I was very curious to see how that cookie will turn out, as its the first one to be so intensely colored. In my opinion, the green hearts looked very beautiful and enhanced the overall impression of the plate. However, the particular taste of pistachio was missing somehow - although I liked the flavor of oranges. Next year I will improve the recipe by adding the particular "pistachio dough" I used to prepare the biscuit of the cake.

I do hope you got inspired by the variety of cookies I presented, and that you are already looking forward preparing them for next Christmas...I am sure your family and friends will very much appreciate your effort.

Yours, Eva