July 31, 2013  |  Education, Flow Cytometry  |  By  |  0 Comments

Cytometry for Interns

As a summer intern at Cytobank, the past few months have been busy and interesting to say the least. I am currently an undergraduate from the University of Redlands in Southern California. Although I have only been exposed to two years of introductory science courses, I have found that not only has it been easy to adjust to the research environment at the Nolan Lab at Stanford and the collegial atmosphere of Cytobank Inc, it has also been remarkably manageable to learn about flow cytometry.

During my two month long internship, I was able to run three different experiments and analyze data for each one. The three experiments were a U937 experiment, a flow PBMC experiment, and the mass cytometry PBMC experiment run on the CyTOF. Below I’ve summarized the flow cytometry PBMC experiment and included some helpful introductory tips I learned along the way – watch for the Mass Cytometry summary in a future post!

Preparing for, Running and Analyzing the PBMC experiment:

The PBMC experiment had five different samples with the following conditions: 1) unstimulated, 2) Human interleukin-10 (IL-10), 3) Human interleukin-2 (IL-2) + Human GM-CSF – (IL-2 + GM-CSF), 4) Phorbol-12-myristate-13-acetate (PMA), 5) Lipopolysaccharide (LPS) from Escherichia coli.

I learned a few organizational skills and tips to run the experiment smoothly. Organizing my materials made it much more simple to follow the protocol. In addition, I discovered that preparing material while waiting for centrifuging steps saved me time and made the experiment run faster.

#1 Be organized

• Make sure to label the samples the easiest way for you to recognize them
• Keeping the samples in (1-5) order in the tube holder trays also simplifies the experiment and saves time

#2 Be efficient but careful

• Do not decant after vortexing, you will then lose all or almost all of your cells
• When adding staining media to the tubes, you can pour to the first line on the tube tray instead of repetitively measuring 4mL of staining media each time

Each of the five stimuli had different concentrations and various antibodies were used for the staining and analysis steps. More details and information for the specific steps is provided here.

The PBMC experimental procedure was straightforward and very easy to follow. I did experience a few surprises:

• When creating antibody cocktails, it’s easier to keep the antibodies in order as shown on the protocol sheet for creating one vial to confirm which one has been used or not to avoid repetition and error.
• Decanting was somewhat of a terrifying experience for me as I thought I was losing all of my cells only to be reassured that they stick to the bottom of the tube.
• Analyzing the data was relatively simple. However, certain aspects were somewhat challenging to a flow novice, such as gating. The helpful and informative tutorials provided through the Cytobank Knowledge Base were great, as they introduced me to the process of gating.

All of the researchers happily and openly answered questions that I had regarding my own experiments and their own projects. It was great to see a lot of the tools and materials that I am familiar with at school being used in the lab and using the skills I have learned so far to conduct lab experiments. Receiving the opportunity to immerse myself in an advanced cytometry lab and working with a startup company has proven to be an intellectually intriguing and utmost rewarding experience. I look forward to maintaining contact with Cytobank, the researchers, and interns in the near future.

-Puneet Sidhu