Cell cycle analysis?

I'm planning an experiment where I can access intracellular cytokines in a specific subregion of the brain in mice. However, this brain region is quite small, maybe 50,000 cells per animal. I know I will need to pool mice but how many would I need to pool? Can I use 500,000 cells? Pooling more than 10 mice wouldn't be feasible.

Thank you!

Hi, I'm doing my first lab internship and I have a little problem, I fixed some fibroblasts with 70% cold ethanol and then I treated with gentian violet to study their morphology. Now I need to count the cells, but there are many of them. Is it possible to solubilize the violet and treat with DAPI? so I could count them faster in ImageJ

I would like to know if it is possible to use antibodies (in this case I would like to use two markers of neutrophils and monocytes such as FITC anti-mouse Ly-6G and FITC anti-mouse Ly-6C) whose application is referred only to cytometry for visualization in confocal microscopy.

Two different cytometers will give same or nearly similar results for activation markers on lymphocytes?

Hi everyone!

I am performing an assay to evaluate CD107a production by CD4+ T cells using Flow Cytometry. Currently, I am stimulating a suspension of tonsillar cells with OKT3 only at 37°C for 3 hours. However, upon analyzing the results, I found that the signal from the unstimulated cells (basal) and stimulated cells are the same (attached is a picture of the corresponding histograms analyzed by FlowJo). Therefore, I think that the stimulation with OKT3 is not effective. I have read that cells can also be stimulated with CD28 in addition to OKT3. Does anyone have experience with this assay that can provide guidance?

Thank you

I am looking into selecting T cells with microbeads for isolation and subsequent adoptive transfer in immunodeficient NRG mice. I am hoping to do positive selection with microbeads. After column selection, I am wondering how the T cells will act in vivo after transfer. Do antibody/microbead-bound T cells act differently or are eliminated in immunodeficient NRG mice?

Hello everyone,

I am new here, and needed as much advice as I can on the following: I am culturing LLC-PK1 cells, which are a spontaneously immortalised porcine cell line. There is a significant difference between the % live cell count of 14%-19%, and the images I have recorded on microscopy. The medium the cell is grown in does permit growth to confluence of the cells, and the cells are immediately read via the BioRad cell counter, with no lag time, but I am so unsure why there is discordance between the Bio-rad reading and the microscopy observations of healthy appearing cells. Please see Image attached. I would appreciate any help with troubleshooting.

Respectfully,

Trecia.

I'm doing my undergraduate research to select CD133+ cells from ovarium cancer cell cultures. I've been trying to use MACS as the selection method (with MS column) and always get a low number of positive cells (around 13,000 to 15,000 cells from 6-9 million cells).

1. Is this a general result for MACS? Or are there any things that I could optimize to get a higher number of CD133+ cells?

2. I assumed the passage number may affect this too (I've been using P12-P13 of SKOV3), is this a correct assumption?

3. I will use the selected cells for ELISA protein analysis, but I still don't know how many cells I need (minimal number) for that purpose. Is the resulting CD133+ cell number mentioned above enough?

Hello, has someone performed a T cell FACS Sorting after lentiviral transduction? We would like to purify those T cells that were transduced using GFP as reporter, and continue expanding the cells for citotoxicity assays. Our cells had a previous purificación step using a magnetic column.

For your attention, thank You ver much!