Dimitri Szymczak, PhD

Analysis tool for direct and sandwich ELISA – convenient and reliable

11/07/2017 By Dimitri Szymczak, PhD Leave a Comment

Many ELISA kits – one analysis tool

Analyte quantification (in pg/ml or ng/ml) such as IL-6, TNFalpha and numerous other proteins is conveniently performed with immunoassays called ELISA kits. At tebu-bio, we not only provide more than 10 000 ELISA kits but we also support researchers through our techserv team who are on-hand to suggest the best kits and to answer any questions.

Being part of that team, I have noticed a number of often asked questions that can be resumed in one : What is the best method for data analysis? [Read more…]

Optimizing the CAS9 messenger RNA for vector-free gene editing

28/06/2017 By Dimitri Szymczak, PhD Leave a Comment

Sequence engineering and chemical modification of CAS9 mRNA

Cas9 Western Blot in CD34+ cells - Trilink poster "“Maximizing Translation of Cas9 mRNA Therapeutics by Sequence Engineering and Chemical Modification”"

Extract from poster “Maximizing Translation of Cas9 mRNA Therapeutics by Sequence Engineering and Chemical Modification”.

For transgenesis and therapeutic perspectives, vector-free delivery of the CAS9 for gene editing is highly preferable. It avoids the risk of hitting the genome due to the vector integration. Directly using CAS9 mRNA is a top option.

Furthermore, as a new promising class of therapeutic biologics, messenger RNA are the subject of numerous studies.

This year, Matthew Porteus’s and Trilink’s teams revealed key and practical results at the ASGC with a poster you can download below.

Notably, there is a large screen of the mRNA CAS9 modifications based on the indel formation. This interesting comparison includes also CAS9 RNP and reveals the high performance of the CleanCap U-depleted 5-moU CAS9 mRNA. We can also note that for the CleanCap CAS9 mRNA HPLC purification is not required.

“Maximizing Translation of Cas9 mRNA Therapeutics by Sequence Engineering and Chemical Modification”
Download your copy of the poster here.

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Stable Cas9-expressing cell lines

21/06/2017 By Dimitri Szymczak, PhD Leave a Comment

Shen et al (Nature Methods, 2017) explore and identify synthetic interactions among 73 cancer-associated genes. To perform their loss of function screen they combined CAS9-expressing cell lines with a sgRNA library of high titer lentiviral particles. Most of these gene interactions were subsequently validated by drug treatment. [Read more…]

New TIGIT:CD155 cell-based assay to restore immune response

23/05/2017 By Dimitri Szymczak, PhD Leave a Comment

TIGIT:CD155/CD112 immune inhibition (in green)

We previously addressed the PD-1:PD-L1/PD-L2 immune inhibition and the corresponding cell-based tools to screen immuno-oncology agents for restoring antitumor immune response. Unfortunately cancer cells have other ways to escape and survive. Let’s look together at another immune inhibition pathway, namely the TIGIT:CD155/CD112.

[Read more…]

Maximize messenger RNA therapeutic activity with innate immune focused approaches

17/05/2017 By Dimitri Szymczak, PhD Leave a Comment

Messenger RNAs – a promising class of new therapeutic biologics

As messenger RNAs (mRNAs) are easier to deliver into cells then plasmids or viral vectors, they are useful for non-dividing cells. Inversely to the vectors, they ensure genome integrity that is recommended for cell therapeutics. mRNA are also well adapted to transient expression as required for cellular reprogramming, gene editing, and vaccines.

You might like to take a look at this poster, presented by Trilink (renowned modified nucleic acid experts) at the Keystone symposia last March (Pattern Recognition Signaling: From Innate Immunity to Inflammatory Disease). They share results on how to optimize messenger RNA for therapeutic activity.

“Innate Immune focused approaches to maximize messenger RNA therapeutic activity”
Download your copy of the poster here.

Proteins with the High Purity (HiP™) label make the difference

07/03/2017 By Dimitri Szymczak, PhD Leave a Comment

The HiP™ (High Purity) distinction by BPS Bioscience starts, of course (as the name says), with a high purity level. But that’s not enough. Such pure proteins may aggregate, which is not compatible with binding assays. Thus, the HiP™ label also demands a low level of aggregation, or even none at all. [Read more…]

New – Continued use immunotherapy cell line expressing recombinant PD-1

03/02/2017 By Dimitri Szymczak, PhD Leave a Comment

PD-1/NFAT-Luciferase reporter jurkat cell line used to monitor the T cell response inhibition via PD-1 interacting with PD-L1/PDL2

PD-1 / NFAT Reporter – Jurkat Cell Line

The PD-1 reporter cell line is a T cell line expressing luciferase under the control of NFAT response elements. The level of the luciferase activity measured with the One-Step luciferase detection system (BPS Bioscience) corresponds to the T cell activation in response to the TCR activator from the target cell.

To escape, the target cell may present PD-L1 or PD-L2 to PD-1. Indeed, the interaction inhibits the T cell activation. It leads to decrease in luciferase activity. [Read more…]

Next generation non-invasive DNA collection device

15/06/2016 By Dimitri Szymczak, PhD Leave a Comment

Drawing blood for DNA collection

DNA is a useful source of information for research and medicine. It starts by collection, following which it is deciphered by a wide range of analysis, from simple genotyping to deep whole genome sequencing.

The most popular way to collect DNA is drawing blood. Not very pleasant for the patient, as we have all noticed, and it also requires an experienced person, a nurse. The blood must be kept cold and DNA extraction should be done not long after the sampling. Still, it brings high yields of up to 30µg od DNA.

Buccal swab-based sampling

Alternatively, saliva sampling can be used to collection DNA. Sampling is painless, but it is still not an obvious choice especially with children. Yields are highly variable, and lower than with blood. Furthermore, kits are quite expensive.

Today, there is a sampling method to easily collect DNA, whilst also providing high yields. It is based on buccal sampling. I invite you to take a look at how it works and the benefits for your studies. [Read more…]

Best methods for Circulation miRNA profiling (cmiRNA)

13/06/2016 By Dimitri Szymczak, PhD Leave a Comment

We need to find biomarkers for prognostic, diagnostic and personalised treatment development. Notably to fight cancers that affect tissues. Since biopsies are invasive, it’s better to look for biomarkers in body fluids. Indeed, a simple blood sample becomes a kind of ‘liquid biopsy’ to reveal tissues affections. For 13 years, increasing interest has been shown for miRNA as biomarkers and it will last for sure. The 2 main reasons are that they are major regulators of cell processes and they are released from tissues into the blood. They are major biomarker candidates in serum and plasma. Thus, these circulating miRNA (cmiRNA) are the best hope for modern medicine. Still, a lot of research has to be done to determine the specific signature for each pathology, and also depending on the patient background. Obviously, cmiRNA profiling is a key step and requires sensitive and reproducible method. Sequencing, qRT-PCR, several kind of microarrays… Let’s explore together what the best approach could be. [Read more…]

Cost-efficient pre-optimized transfection reagents

10/06/2016 By Dimitri Szymczak, PhD Leave a Comment

The main challenge when choosing a transfection reagent is that we don’t know how it will work with our own cell type of interest. It is also time consuming to find the optimal conditions. Well, here’s the solution: � pre-optimised transfection reagents.

eGFP expression revealing transfection efficiency

Pre-optimised transfection of pEGFP-N3 plasmid expressing eGFP under CMV promoter

They already cover more than 39 cell types including MEF, Caco-2, MCF-7, HepG2, Primary Macrophages, Huh-7 and many others.

Sharing our feedback on performances

Using a vector expressing the eGFP (pEGFP-N3) under CMV promotor, we assessed the transfection efficiency. Take a look at the example of results below. They will give you a pretty good idea of what you can expect.

Specific feedback of Genjet transfection

Transfection efficiencies with pre-optimised Genjet reagents

Can we compare the pre-optimised Genjet?

Ok, now you can see we got good feedback. Still, will it be a better solution?

The answer is yes. The comparison with Lipofectamines, Fugene HD and Amaxa reveals that� pre-optimised Genjet reagents allow high number of positive cells (dark green below).

You can see on right of each picture just above, that performances are even better with 10% serum, that would be much appreciated by the cells.

Furthermore, this quality is associated to low price. Check it for yourself!