Is there a cell fixation protocol that won't affect the RNA or the cell surface markers?

More Lina Ghezil's questions See All

How can the modification of garden soil cause global warming?

Hello, I am working on a soil project and I found a primary research article that tests how the modification of agricultural soil can cause global warming, using a thermal collector, however I...

02 March 2021 7,161 3 View

How to use the bioluminescent ATP assay kit?

I need to do a mitochondrial ATP quantification assay in insects and for this, I have a bioluminescent ATP assay kit but I don't have a protocol yet. I'd like to know if you have some kind of...

24 January 2021 3,655 3 View

How to find solar heat flux to assign in fluent boundary?

Hi all researcher, I needs help to determine solar heat flux if I have a value of solar radiation (w/m2) and solar absorptivity coefficient (alpha). Does it it have any formula? I want to assign...

22 October 2020 1,977 5 View

Could anyone recommend a scale for measuring Dweck's Growth Mindset?

I intend to conduct a study based on Dweck's theory of mindsets (implicit personality theories, or self-theories). I am looking for a survey for measuring a mindset for employees in organizations.

10 August 2020 4,200 6 View

How much heat transfer coefficient of air?

I am looking of value heat transfer coefficient to assign in Ansys steady state thermal simulation. But I'm not clear about heat transfer coefficient is for material or air? Because in...

20 January 2020 7,342 5 View

Western Blott results?

To publish a result of western blott it is necessary to show the bands of the total protein? In the case of not showing this part of the result, can it still be published?

20 March 2019 5,596 2 View

What topic should i focus on my MSc accounting and finance dissertation proposal?

Hi.. I have some topics I'd like to focus on but still debating which to focus on and whether they have a lot of background to evaluate. The following topics: -Accounting ethics and sustainability...

12 December 2018 4,127 3 View

What is the best method to analyse the mediator?

I have a framework which contains: 2 independent factors with 5 likert scale response psychosocial factors (3 domains) physical demands (3 domains) 1 dependent factor with yes/no scale 1...

25 July 2018 3,671 6 View

Usually what is the format (order of the content)to be used for research reports which is to be submitted to the government for policy making?

I am doing a project for the Government for the first time. Could somebody help me with the format to be used for the same. I see a lot of policy researches that are done without following the...

29 June 2018 1,299 5 View

If i want to do a flowcytometry experiment, when should i incubate my cells with ligand? before or after deataching the cells?

i am transfecting adhernet HEK 293 with flourescently tagged nuclear receptor and i want to see the effect of ligand treatment

19 June 2018 5,192 5 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 View

No title

02 March 2021 3,060 3 View

How do I determine the degree of polymerization of PEG 400 based on NMR integration?

Hello, I am working on the photocatalytic degradation of PEG 400. I would like to know if it's possible to determine the degree on polymerization based on NMR integration, or by the MW of the...

02 March 2021 6,186 3 View

Do you know any MR image dataset?

I would like to research on MR images (0.5T and 3T). Can you please suggest some websites that I can download dataset including both 0.5T and 3T MR images? Thank you.

02 March 2021 7,735 3 View

Energy minimization in LAMMPS using NVT ?

How do I do energy minimization in lammps with NVT ensemble ? I am using the following command and it does not seem to work. It stops at extremely high energy and gives me the error zero search...

01 March 2021 2,294 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

How can I edit suggested publications?

Hi there! I regularly receive suggestion about papers and authors that are totally out of my area. Does anybody know how I can fix this? Thank you!

01 March 2021 9,574 4 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Henry Kolge

Paraformaldehyde can be one of the fixative. Its percentage can be altered between 1-4 %

I have used for fixing cells to be used in FACS, Fluorimetry, confocal microscopy etc

Anjali Srivastava

It is challenging to find a cell fixation protocol that will preserve both RNA and cell surface markers, as fixation can potentially affect the integrity of both. However, there are several options that can be optimized to achieve the best preservation of both.

One potential approach is to use a fixation method that is mild and fast, such as paraformaldehyde (PFA) fixation for a short duration. This can help to preserve the RNA and cell surface markers, but optimization of the protocol is essential.

Here are some general guidelines for optimizing a PFA fixation protocol:

Use fresh PFA: Paraformaldehyde should be freshly prepared or thawed from a frozen stock before use.

Optimize fixation duration: Determine the optimal fixation time for your specific cell type and surface markers. Fixation times can vary from a few minutes to several hours, depending on the cell type and the target antigens. For example, in general, a 10-minute fixation with 1% PFA at room temperature is a good starting point.

Use gentle fixation conditions: Use mild conditions that are compatible with preserving RNA and cell surface markers. For example, a mild buffer (such as phosphate-buffered saline, PBS) can be used for washing and diluting the PFA solution.

Optimize antibody staining: Use primary and secondary antibodies that are optimized for PFA-fixed cells, and consider using permeabilization to allow the antibodies to access intracellular targets.

Check fixation quality: To confirm that the fixation protocol is working optimally, perform quality control tests such as flow cytometry or immunofluorescence microscopy.

It is essential to note that optimization is critical for preserving RNA and cell surface markers, and different cell types and targets may require different fixation protocols. Therefore, it is recommended to perform optimization experiments for your specific cell type and surface markers.