Kun Yun

Mayo Clinic - Rochester · Department of Molecular Medicine

Hi, I did some scRNAseq as well as protein expression analysis on the same sample, however, the RNA level is not consistent with the protein level. How do people usually explain this piece of data in manuscripts? Thanks!

Hi, I'm using a very hydrophobic peptide for tetramer staining. The peptide can only be dissolved in DMSO, and then further diluted by PBS or water. I have tried 10, 5, 3 and 2 mg/mL as the stock concentration, and only when it's dissolved at 2 mg/mL in DMSO, it can be further diluted in PBS or water without any aggregation.

However, for tetramer staining, it's suggested to not exceed the final concentration of 10% DMSO in the peptide exchange reaction for tetramer assembly, but in my case, the 2 mg/mL peptide stock leads to a 16% DMSO in the exchange reaction. Does anyone have any experience to somehow lower the DMSO concentration in the reaction? Like add more monomers or add more PBS to the reaction to increase the final volume?

Thanks!

Hi, I have been trying to dissolve a very hydrophobic peptide (containing cysteine) with DMF, however, the solution looks very cloudy when I add DMF, and further dilutions with water doesn't help much. Does anyone have any tips on this issue? In addition, I'm use NanoDrop to measure concentration, but when I use DMF as blank, it shows "bad blank". By the way, I'm measuring the concentration at A280. Thanks!

Hi, I'm trying to block FcR on human macrophages in some in vitro cocultures with human T cells. Is there any FcR blocking reagents used in vitro culture?

Thanks,

Kun

Hi,

I have a basic question about cell engineering. Is it possible to load or engineer mammalian cells to produce certain chemicals?

Hi,

How long do you usually rest cryopreserved dendritic cells after thawing? And what cell culture media does it need for resting? Thanks!

Hi,

Has anybody ever seen stained cell population moves downwards compared to FMO?

Hi,

I'm trying to stimulate human T cells with magnetic beads coated with antibodies. I have streptavidin-coated beads as well as biotinylated antibodies. According to the vendor, they usually incubate cells with antibodies first and then mix them with beads. I'm wondering if it's feasible to immobilize the antibodies onto beads first and then add to cells. Thanks!

Hi,

I'm planning to electroporate mature dendritic cells with in vitro transcribed mRNA, but I'm not sure how people usually decide the dose of mRNA and number of mDC to reach a good transfection efficiency (80-90% positivity). Does the total amount of RNA or the concentration of RNA matter? Thanks!

Kun

Hi,

I have been using HighScribe T7 polymerase kit for in vitro transcription from NEB, but the protocol gives a 20uL reaction with 1ug DNA template, which is not enough for my downstream experiments. Does anybody know if I can just proportionally scale up all the reagents or do I have to divide into multiple 20uL reactions to reach the amount of RNA I need? Thanks!

Kun

Hi, does anyone know how to find the DNA sequence encoding the scFv region of a specific antibody? Is there a library for these? Thanks!

Hi, I'm using neutralizing monoclonal antibody to neutralize cytokine in cell culture. I added neutralizing antibody on Day 0, and measured the cytokine level 3 days later, but it seems that the cytokine level is still high. Is it possible that the neutralizing antibody is degraded in cell culture within 3 days? Thank you!

I read papers about in vitro transcribed RNA. What is the difference between in vitro transcribed RNA and commercially purchased RNA?

Thanks

I am trying to expand CTLs in vitro and have noticed that many studies usually use irradiated PBMCs as feeder cells besides supplement with cytokines. What ratio of CTL to PBMCs does it usually require to reach a decent level of proliferation?

Thanks!

Hi,

I'm activating CD8 T cells by adding MHC-I restricted epitopes to whole PBMCs in vitro according to previous published studies. I noticed that cell feeding occurs 24 hrs after adding peptides and the feeding procedure involves resuspending cells by pipetting gently. Does this disrupt the interactions between DC and CD8 T cells and negatively affect T cell activation? Thanks!