A good method to purify monocytes for culture from sepsis patient's blood

I have collected PBMCs from blood samples and frozen them. Now I have to thaw them and use monocytes.

Does anyone have a protocol for thawing and monocyte adhesion from these frozen PBMC samples?

Monocyte isolation is a time and cost comsuming procedure. Does cryopreservation change the characteristics of monocyte in term of immune responses?

I am currently working with human monocyte/macrophage cell lines but want to extend my research to short term cultures of monocytes from human blood. Does anyone have a reliable method to prepare PBMC and purify monocytes for culture.

Hi ResearchGate community,

I have been trying to learn more about the optical differences between block-based real-time PCR machines like ABI StepOne versus rotor-based machines such as MIC or RotorGene systems.

I understand that some systems rely on ROX as a passive reference dye while others state that it is optional to incorporate it and others do not need such a factor at all.

My question is if you add this fluorescent dye to your master mix, would it interfere with the detection when it is being amplified using one of the systems that do not need such normalization?

Highly appreciate any insight in this regard.

Best,

Negar

Hi everyone, I´m working with A549 cell line and I am going to use TRIZOL for RNA extration.

Can someone help me with some advices to extract RNA from this type of cells?

Thanks!

I would like to know how I can tell when fluorophores will or won't work when it comes to flow cytometry. I know some basics such as you can't use colors that are excited by the same laser and pass through the same filter. Recently, I did a flow experiment where I used PerCP-Cy5.5 and Brilliant Violet 711 in my same panel. I thought it would work but the spectral overlap was over 100% and I now realize that is probably due to them passing through the same filter on our facility's cytometer (even though they are excited by different lasers and detected by different detectors).

I also once used APC and Alexa Fluor 700 together and got a spectral overlap warning of over 100%. Unlike the previous example, these colors are excited by the same laser but pass through different filters and are detected by different detectors.

These situations leave me a bit confused as to how I can tell when fluorophores will work in my panel or not. In general, now I am trying to craft panels where colors pass through different filters and detectors regardless if they are excited by the same laser or not. And it seems like regardless of anything I do, I can't use more than one color excited by a 640 nm laser (i.e. APC, AF647, AF700, APC-Cy7 etc.) or beyond as they always seem unhappy together.

I am extracting miRNA-enriched RNA with miRNeasy Serum/Plasma Kit (QIAGEN #217184) and the addition of TRIZOL and cloroform before the centrifugation at 4ºC made me wonder if these reactives have to be at room temperature or they should be at 4ºC.

Thanks!

I'm trying to measure the ROS production from RAW 264.7 cells by using DCFH staining and analyst by flow cytrometry. The problem is the level of ROS production in control cells was high. I'm not sure what am I doing wrong. Can anyone help me solve this problem?

Kindly confirm how can we interpret data of delta Ct in RT-PCR Results?

We have just control tissue, not any test sample; thereby delta delta Ct values can't be find out.

We hv just delta Ct values (house keeping and test gene of control tissue only).

How to interpret the data with delta Ct values only ?