What will happen If a transgenic mouse expresses transgenic TCRs that recognize an antigen of E. Coli presented by an MHC-class II allele?

I am looking at puromycin incorporation rate by flow cytometry using a specific anti-puro-antibody which I use to stain different cell types. For some cells the signal is high (i.e. Puro MFI), while for other cells the MFI is negative!!

Would that be a problem of scaling the values for theis marker or adjusting the voltages of the PMTs on the cytometer?

Thank you for any advice or assistance you may offer.

I will be assisting my colleague with MCF7 subculture and later performing MTT assay. We will be using DMEM media (with phenol red) along with 10% FBS and 1% penicillin/streptomycin. We will revive the cells after procuring the cells from the supplier. We plan to passage the cells twice and perform the MTT assay in the third passage. Is a third passage fine or I need to passage the cells few more times?

Thank you in advance for your replies.

My purpose is to do single nuclei RNA-seq. I have two samples: young mouse and aging mouse.

The procedure is like this:

1. Use Collagenase P and Disparse II enzymes to digest joints, followed by DnaseI dissociation, to get single-cell suspension.

2. Follow the protocol Detergent-mechanical Cell Lysis,  Homogenization and Sucrose Density Gradient main steps to get single nuclei.

Then I began step 1 by following the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) protocol.

The question is:

1. after finishing step1 of v3.1 protocol, the aging mouse didn't form homogeneous solution? I think it's called wetting failure.

2. After step2 cDNA amplification, the cDNA QC of young mouse has some problems.

First, the concentration is not high. Second, the cDNA number within 200 -9000 bp is significantly lower than 95%

Could anyone help to give some opinions?

I opted bodipy due to its smaller stoke’s shift (493/503) but my images do not have consistent results. in the same well few fields are clean without background and other fields have background. It looks where the cells are in group in the co-culture astrocyte and neurons there is no background. these cells are plated and fixed in 96 well and imaged in opera Phenix.

I

Is tRNA necessary for the extraction of DNA with magnetic beads? If does, why? And also the glycogen.

I am currently trying to understand the RNA extraction procedure. We used a plant RNA extraction kit (the E.Z.N.A plant RNA extraction kit from omega bio-tek) that involved cell lysis with a homogenizer column, addition of ethanol and then an RNA mini column that binds the RNA, and then we eluted the RNA using DEPC water.

What I want to know is how the RNA mini column only binds to RNA and not DNA? Is it something to do with the buffers having DNases so the DNA is degraded, or the column deferentially binds to RNA over DNA? Or does this depend on the kit used as to how only RNA is extracted?

Any insight would be extremely appreciated.

I want to why their is difference in pH of tris in resolving gel 8.8 and in stacking gel 6.8 during SDS - PAGE in western blotting, with having all the composition same in making of gels ?

Hi, everyone.

When I do flowcytometry analysis and calculate mean fluorescence intensity (MFI), I sometimes find the values are "negative".

Does that mean the staining was inappropriate or the intensity was nearly zero??

It would be fine if someone could tell me what does it mean.

Thank you in advance.

When I run the assay as singleplex I get great results; great PCR efficiency. When I perform a duplex assay my CT(s) are similar to singleplex assay  for HEX label probe, but not for the FAM label probe.  My Ct(s) are quite higher for FAM labeled probe in the multiplex assay compare to singleplex assay.  For example, in the duplex assay my Ct is 21.2 and in the singleplex assay my Ct is 38.2.  

The  frustrating part is my standards curves for everything is great. My Ct(s) values for duplex assay are similiar to the Ct values for the singleplex assay for both probes (FAM and HEX).  I am using a plasmid as my standard.  The problem comes when I used actually samples; the FAM labeled probe for the duplex assay is giving me false positive results.

What is going wrong here?  Is there cross talk between probes ? Is Hex probe interfere with FAM? Should I use a different probe like FAM and VIC? Is it the mastermix ?  I am currently using Bio-Rad SsoAdvanced Universial probe SuperMix. or Should I alter the primers concentration?