As we run tests and learn new information in our lab, we like to pass our findings along to our customers so they can get the most from our immune cell products. When we get questions from curious scientists, we head to the lab to try to find answers and publish those findings for everyone to use.
Several customers have recently asked if we have validated our recall antigen assay. While we have not pursued a formal validation, we have examined the longitudinal data of our donors and are happy to report that the results of our recall antigen assay are fairly consistent over time.
The following are just two examples of what we have gleaned from running this assay consistently on our PBMC.
Longitudinal Recall Antigen Testing
As seen in the graph below, PBMC from donor 369 produced IFNγ when stimulated with either tetanus toxoid or CMV antigen. The responses have been relatively steady since May 2017. A pipetting error may have caused the large standard deviation observed in July 2017. PBMC collected in February 2017 had much lower cytokine output, which raises questions as to what may have affected the cells at that time.
Looking at the recall responses over time for another donor in the graph below, we found that PBMC from donor 290 were positive for CMV response as measured by TNFα production. However, donor 290 produced little to no response to TNFα when stimulated with tetanus toxoid. The low response does not indicate that donor 290 is unresponsive to tetanus toxoid because there was plenty of IFNγ found in the sample.
We have noticed that some donors produce TNFα in response to tetanus toxoid or CMV while others do not. There is also a tendency for donors to produce higher (>4,000 pg/mL) or lower (<2,000 pg/mL) TNFα following stimulation with LPS, a substance known to activate monocytes.
TNFα levels do not necessarily relate to the percentage of monocytes present; it seems to be a donor-specific characteristic.
A Note on Culture Media: Alternatives to X-VIVO™ 15
Many people have asked if they can use RPMI 1640 supplemented with serum instead of the serum-free X-VIVO™ 15 medium we mention in our protocols, especially since X-VIVO™ 15 is unavailable at the time of this writing. We have been testing various alternative media and will publish those results later this year.
For a sneak peek of our findings, see the two graphs below, which show a comparison of DME/F12, IMDM, and RPMI 1640, each supplemented with 10% FBS. We used X-VIVO™ 15 as a control and ran our standard recall assay to measure IFNγ and TNFα. The use of FBS clearly increased the amount of both cytokines in the control condition.
DME/F12 seems to be the best choice for detecting IFNg production with the caveat that X-VIVO™ 15 was superior with LPS. RPMI and X-VIVO™ 15 both supported greater TNFα production with the exception of CMV antigen stimulation, which seems to benefit from the DME/F12 medium.
Based on this short-term assay, we recommend DME/F12 with 10% FBS as the best choice to replace X-VIVO™ 15. We will continue to run more extensive experiments to find the best medium and supplementation and will post our findings when available.
For more on this topic, read our case study:Learn More