Does anyone have good protocol for dissociation of hippocampal neurons from adult mouse (flow cytometry/RNA seq)?

More Yuka Obayashi's questions See All

Are there any reliable antibodies for excitatory (glutamatergic) and inhibitory (GABAnergic) neurons in hippocampus?

I have Camk2a-Cre mice model, and would like to have antibodies for excitatory (glutamatergic) and inhibitory (GABAnergic) neurons in hippocampus CA1 regions. Do you know any good antibodies for...

10 November 2018 9,467 4 View

Any (hippocampal) culture and cell line useful to investigate epigenetic regulation (ie. high proliferation rate)?

I am currently looking for (preferably hippocampus) culture/neurosphere/cell line with high proliferation rate, which are available to purchase in Canada.

01 February 2018 4,687 2 View

Do you have any suggestions for neurosphere assay protocol, using adult mice?

Do you have any suggestions for growth factors (EGF and FGF) effective/useful for proliferation medium? How do you sterilize surgical equipments - autoclave or buffer? Any tips to share -...

01 February 2018 1,114 2 View

Problems in using mouse host primary antibodies on mouse brain sample (IF/free floating staining).

I am doing free floating immunofluorescent staining on my mouse brain sample. I've been having difficulty using rodent host (ie. mouse, rat, guinea pig) primary antibodies, and I don't know why....

01 January 1970 6,925 13 View

Having difficulty IF staining on 5XFAD mouse brain sample (free floating).

I am not sure if we are supposed to change staining protocol for different mouse strains, but I am having difficulty staining (free floating immunofluorescent staining) on my 5XFAD mouse brain...

01 January 1970 3,227 5 View

Are there any rodent behavioural experiments that can substitute contextual fear conditioning test?

I am investigating the role of histone modification during hippocampal memory formation. It is the best if I could perform contextual fear conditioning on mice. However, we unfortunately do not...

01 January 1970 9,449 1 View

Reversal learning deficit, but normal memory extinction on Morris water maze test

I am wondering if anyone has seen the results of morris water maze like this; - normal acquisition (decrease in escape latency) BUT - significant deficit in reversal learning - normal memory...

01 January 1970 8,033 4 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

What do I do with DLS peaks that are from the buffer?

I'm looking at the aggregation of my protein sample using DLS. Unfortunately, my buffer (20mM HEPES) also results in a set of peaks. These are at approximately 1 and 1000 d.nm. The lowest peak...

01 March 2021 9,015 2 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

I have two groups of brain samples, control and treated for example. It was total RNA nova seq sequencing. I tried all the available pipeline like: star+rsem+deseq2, Hista+stringtie+cuffdiff,...

27 February 2021 356 6 View

Can Ethovision store the generated data and video recordings directly to NAS system?

The size of data generated by Ethovision is huge. Having NAS system for data storage seems to be a good option, I am wondering whether Ethovision can directly store the data to the NAS system...

27 February 2021 6,350 2 View

Why is the efficiency of viral mRNA electroporation in PBMC low?

Hello, I have been struggling with the electroporation of PBMC with HIV-I mRNA. Although the quality of the RNA generated is good, and the handling of the cells in meticulous, the frequency of...

27 February 2021 9,233 3 View

Why initial concentration of Pb not the same as calculated one when testing using AAS?

I am currently doing research on the adsorption of heavy metal using magnetic reduce graphene oxide. I use AAS to determine the concentration of the heavy metal. I prepare the Pb solution with...

26 February 2021 9,580 4 View

Is there anyone who experienced with cytotoxic effect of Hoescht 33342 dye?

Is Hoechst toxic to cells? In general, it is known as non-toxic. I am working with dsRED MDA-MB-231 cell line. I applied it for my PDMS-based microfluidic platform, for this reason, I incubated...

26 February 2021 9,119 3 View

How to handle studies which report perfect effect sizes in a meta-analysis using Pearson's r?

I'm in the process of updating a meta-analysis and in one of the new studies, effect sizes are reported as Tau-U. Two of the effect sizes are reported as Tau-U = 1.00. I am using Andy Field's...

24 February 2021 2,265 3 View

What is the best concentration of cDNA to be used for qPCR?

I have synthesised cDNA from 500ng/ul of RNA. The concentration of the cDNA Is about 5000ng/ul. Should I dilute my cDNA or just use it as it is for my qPCR? I usually use 2ul of cDNA for a 20ul...

24 February 2021 8,618 3 View

Bahman Sadeghi

Please check the following paper in which investigated the effect of CA1 and CA3 pyramidal neuron dissociation on brain transcriptomes:

Article Hippocampal transcriptomic responses to enzyme‐mediated cell...

But if you are looking for a full protocol from a to z, that includes necessary reagents, materials as well as how to harvest adult mice brains, hippocampus dissection, homogenization, myelin removal,..., to FACS, RNA seq, etc. check the following:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326170/

Article RNA Sequencing and Analysis

Article A practical guide to single-cell RNA-sequencing for biomedic...

Yuka Obayashi

Bahman Sadeghi Thank you very much for valuable information!