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Questions related from Theodore Kapellos
I am doing reverse transcription using a semi-suppressive PCR therefore I introduce a template switching oligo at the 5' end of my transcript which is complementary to the PCR handle at the 3'...
10 October 2018 7,665 2 View
I want to carry out a qPCR for a human gene of interest and I need to decide on a suitable reference gene. For the past few years I have been using Actg1 for my murine studies and therefore I...
07 July 2014 1,712 7 View
I want to stimulate a murine endothelial cell line (sEnd.1) and test my drugs of interest to quantify anti-inflammatory potential. It is accepted for endothelial cells to be treated with TNF-α...
12 December 2013 4,814 9 View
A question of cytokine kinetics. In an activation assay, we usually want to measure effects of our treatment (whatever this might be) on pro- or anti-inflammatory cytokines. Most commonly...
11 November 2013 7,843 4 View
Do you culture for 6 or 7 days, do you plate a certain number of cells on Petri dishes, do you wash the media on day 3?
11 November 2013 4,550 12 View
I homogenized murine lungs in 0.5% w/v HDAB in 50mM sodium phosphate buffer to perform a MPO activity assay. The protocol I do have instructs that I should use 20ul of my samples with 160ul TMB...
10 October 2013 2,176 4 View
I culture BMDM from young male mice. I came across a Science paper where circadian oscillations in several pro-inflammatory markers as well as monocyte differential behaviour was highlighted. So,...
10 October 2013 3,728 1 View
I finished an ELISA assay and read it to discover two hours later that I saved the 630nm data which I used to correct my 450nm data. I immediately measured the plate again but I'm not sure whether...
09 September 2013 1,435 20 View
I have some qPCR data which I want to represent in graphs. I used to use the DCt values but the problem is that they are reciprocal to the expression changes (high DCt values means low expression)...
07 July 2013 465 2 View
I want to design qPCR primers for eGFP so I've looked up the literature for its DNA sequence but the outcome was rather confusing; too many "GFP" sequences that I now don't know which one is the...
07 July 2013 4,723 2 View
I am stimulating microglial cell lines (BV-2 cells) with LPS and IFN-γ. I want to study how inflammation develops in these cells and therefore want to investigate the pathways activated early in...
06 June 2013 8,094 35 View
I want to inject i.p. Annexin or Dexamethasone to mice for the needs of my in vivo experiments. After these injections I will administer inflammatory mediators and I want to see whether...
05 May 2013 4,180 23 View
Do cannabinoids have a trancription-specific effect only or do they also affect other processes in the cell?
04 April 2013 8,263 8 View
We recently purchased a canabinoid receptor KO mouse strain to use for our experiments. Although genotyping so far showed that the mice were true KOs, when we isolated cells from the BM of these...
03 March 2013 6,506 26 View
I want to set up an endotoxemia in vivo model and I plan to use i.p. injections of LPS from E. coli to induce the disease. I ordered the O127:B8 E. coli serotype LPS, but I found an unopened vial...
03 March 2013 1,712 6 View
I tried to measure IL1β levels secreted by 1ug/mlLPS or 500ng/ml IFN-γ BV-2 cells and I didn't find any in the medium. My dilution was 20x but I'm worried that this cytokine is not cleaved (and...
02 February 2013 4,822 7 View
What is the point of filtering FBS/FCS before applying the cells to media and after heat inactivation? Is it just to remove small particles/debris or for the removal of microorganisms, as well?...
12 December 2012 5,548 12 View
I want to set up an inflammation cell culture system to study the effects of an anti-inflammatory agent developed in my lab. For positive control, I was thinking of glycocorticoids (e.g. Dex,...
11 November 2012 4,650 31 View
Short question: why does one add PBS+EDTA in adherent cell cultures to detach them? And is it more preferable than trypsinization?
10 October 2012 438 9 View
I did a lab designed ELISA for OVA-specific IgE levels measurement in mouse serum. The results are as one would expect, however the standard curve failed completely (except for BLANK); it didn t...
08 August 2012 786 2 View
I designed a primer pair for qPCR and looked for melting temperatures between 52-58C. Eventually I had 57.3C and 58.8C. I then tested the specificity of my primers by using a gradient PCR and a...
06 June 2012 5,083 15 View
When one is designing primers for a PCR, what E-values should expect when primers do not anneal to random sequences in the genome? My primer set has an E-value of 0.013 for the gene I wish, and...
04 April 2012 1,971 5 View
I have an argument with colleagues and they tend to answer by experience, not by logical conclusions. How can a lyophilised powder, e.g. BSA, OVA, etc. be non-sterile? My colleagues argue that...
03 March 2012 2,695 5 View
I have been using two different protocols for cytokine intracellular staining and getting different results. I used eBioscience and BD Bioscience. The first protocol did extracellular staining...
03 March 2012 5,879 5 View
I have been doing sandwich ELISAs the last week but I ran out of the Costar ELISA plates I had been using and used anoher company's (Greiner One). Both plates were flat bottomed 96 well plates....
02 February 2012 2,983 22 View
I want to study naive CD4 T cells polarization and therefore I need to be sure that the cells I will culture will be naive. So far I have found isolating kits from Miltenyi, STEMCELLS Technologies...
11 November 2011 9,610 7 View
Hi everyone, I am applying RT-PCR using SYBR GREEN and i am really worried whether I have contamination in my samples or not. This is why I also run no RT controls and a NTC for both my GOI and...
06 June 2011 9,889 2 View
