Tetanus Toxoid

Dear colleagues, 

I have a very silly question, but it bothers me a lot. 

When working on cell viabiity using a hemocytometer, how do you account the cells that were already dead and had detached BEFORE you trypsinized the cells?

Most protocols I saw insist that one should always aspirate medium before trypsinizing. But it means that a significant portion of the dead cells will be out of the assay due to aspiration. 

I'm currently in the process of immunofluorescent staining H9 and Jurkat cells, but I'm having trouble with staining these cells. Our difficulties arises from the antibodies not fluorescing. I've tried several troubleshooting methods including increasing detergent concentration to promote permeability of the cells -- this has not worked. I've also used different slides such as gelatin coated slides. I've increased antibody incubation times but this hasn't helped either with the staining. Although the antibodies haven't been working properly, DAPI staining has produced nice nuclear staining. 

A protocol for the current procedure I've been using is attached below. 

Thanks 

Do we have a center in the Philippines that perform TUNEL Assay using lactic acid bacteria? 

Dear Colleagues!

Recently I've performed TEM observation on cultured spermatogonial cells. I've found two unknown structures (attached below). I thought, they're just artifacts, but they reappeared several times during my observations. I tried to find analogical structures elsewhere, but to no avail. Does anyone knows what could it be? I'll be grateful for any advice.

I am using MCEC (Mouse cardiac Endothelial cell line) and using Methylglyoxal as agonist. I would like to know what could be possible ways to explain this phenomenon. I have also tested and it is not an artifact as agonist is not interacting with FURA dye. 

I am going over a reviewer's comments for my paper and being new to the research world I am not sure what the reviewer means when he/she is asking me whether the inhibitory effect of my product can be "rescued" for my cancer cell growth. He says I should do this to rule out toxicity. I did an MTT test, which did not show any effect of my product on the cell viability. Thanks in advance!

We always use HEK293A cells to package adenvirus, could HEK293T cells be used to do it?

Thank you~~~