A Tale of Leishmania Immunity in Multiple NLRP10 Knockout Mice

Congratulations to my friend and co-worker for many years, Gwen Clay.   This week an interesting study was published in the Journal of Immunology evaluating the role of a protein in the NOD-like receptor family (NLRP10) during cutaneous leishmaniasis.  Gwen and I had countless discussions about data interpretation that were made all the more complicated upon the discovery that the first transgenic line harbored a second mutation (DOCK8).  Thanks also go out to the many co-authors on this paper, especially Mary and Diogo, that saw this study through to its conclusion by performing the necessary follow-up experiments in additional lines of mice.

 

HS029 – Various Culturing Conditions for L. tarentolae

Submitted by Hannah Sparks

A new culture of L. tarentolae was seeded due to the culture results showed in HS026 that 1/2 X BHI of L. tarentolae died due to insufficient nutrients. This culture will be seeded in 5% FBS 1X BHI and 1% FBS 1X BHI. The media should provide sufficient nutrients for the L.Tarentolae to survive on. Once checked under the microscope, the parasites showed signs of being healthy. We expect the growth rate to be faster than the previous culture and for the parasites to be healthier.

HS029

 

HS023 – Mysterious swollen L. tarentolae (or budding yeast?)

Submitted by Hannah Sparks

This experiment is to perform a count of L. tarentolae that was made by B. Crane (refer to BC011). Upon initial observation, the parasites seemed to be lethargic and round. It appeared that they might be budding yeast due to the daughter cells budding from the parent cells were smaller. In order to determine if they were budding yeast or parasites, I aspirated the media from the Flask and added fresh media and let them sit in the incubator overnight. The next time they were observed they still did not appear to be normal parasites.  However, due to the appearance that they might have flagella, we were unable to conclude that they were budding yeast. Dr. Graff also reasoned that the parasites might have been swollen due to hypotonic conditions where the BHI being only half as concentrated as normal.

023

 

 

HS020 – Continued attempts at slowing the growth rate of L. tarentolae

Submitted by Hannah Sparks

This experiment was to slow the growth rate of L. Tarentolae. Dr. Graff aspirated the media from one flask with 1X BHI and one flask with 1X BHI with 1% FBS and added media to both flasks again. The parasites grew remarkably fast again, so we decided to reduce the concentration of the BHI to half. Results showed that the parasites grew too quickly again, so we concluded that it would be best to start a new culture altogether. We may reduce the concentration of the BHI to 0.2X as an additional measure to slow the growth. 

HS020

BC013 – Starting a culture of HEK293 cells

Submitted by Brittnee Crane

I taught Luke Dominico how to start a culture of HEK-293 cells. A frozen aliquot, from the -80°C, was used to seed the culture. My intentions are to treat the cells with LPS and 24 hours after the LPS treatment, to obtain the mRNA and purify it. The mRNA will be put through an RT reaction intended to produce cDNA. The cDNA will then be used in PCRs.

BC013

BC012 – Starting a new culture of THP-1 cells

Submitted by Brittnee Crane

I started a culture of THP-1 cells, using a frozen aliquot from the -80°C. The aliquot used, was from a batch that I grew and froze back, in what I found to be ideal conditions. My intentions are to treat the cells with LPS and 24 hours after the LPS treatment, to obtain the mRNA and purify it. The mRNA will be put through an RT reaction intended to produce cDNA. The cDNA will then be used in TRIM PCRs. Previous studies have shown that TRIM proteins are expressed at different levels in THP-1 cells.

 

BC012