Amanda Rocha

20 Questions 9 Answers 0 Followers

Questions related from Amanda Rocha

Someone recommend to use CFSE to stain parasites as Trypanosoma or Leishmania species to detect cells infection?

I have seen that some groups use CSFE to stain some parasite species before infect cells to detect percetage of infected cells. Could I use CSFE to stain Trypanosoma cruzi and measure infection in...

16 August 2020 2,054 2 View

Why mercaptoethanol in THP-1 culture medium?

I have purchased THP-1 cell line from ATCC and they recommed mercaptoethanol. It is a reducing agent, what is the function of this kind of agent in culture medium of THP-1 cells? How...

29 May 2019 8,255 7 View

What is recommended temperature to perform fixation with formaldeihyde or paraformalde to intracellular staining by flow cytometry of culture cells??

Is it best perform fixation step with formaldeihyde or paraformaldeihyde at 4ºC or room temperature or there is no great difference? I work with human mo-DCs in culture and I need to detect...

13 May 2019 3,661 3 View

What is the difference between formaldehyde and paraformaldehyde in cells fixation for intracellular staining using flow citometry?

I need to detect and measure the abundance of cytoplasmatic proteins (AIM2, IFI16, GBP1, GBP4) using direct detection by flow cytometry with fluorochrome-conjugated antibodies in human mo-DCs and...

12 May 2019 1,620 4 View

What is the best permeabilizing agent to detect soluble cytoplasmatic and nucleic proteins by flow cytometry?

I need to detect and measure the abundance of cytoplasmatic proteins (AIM2, GBP1, GBP4) and some nucleic proteins (IFI16)using direct detection by flow cytometry with fluorochrome-conjugated...

12 May 2019 9,177 2 View

How many human primary cells I need to detect caspase 1 in western blot?

I want to detect cleaved caspase 1 in human monocyte-derived dendritic cells after infection with my pathogen

15 January 2019 4,655 5 View

Which I prefer to promote knockdown of a gene in human monocytes-derived dendritic cells: siRNA or morpholino?

Initially, I have thought to use siRNA to study the role of specific genes in mo-DCs infected with T.cruzi, but I found this work these days:Induction of the interferon response by siRNA is cell...

18 December 2018 850 3 View

What plasmid is recommended to knockout genes in human cells using CRISPR/Cas9?

I have pX330 available in my lab, but I haven't found many recent works using this plasmid.

11 November 2018 9,407 3 View

My targets amplified in these different RNA input?

I have tried to established the amount of RNA input in my RT-qPCRs reactions to amplify interest targets in human monocytes-derived dendritic cells and I perform a RT-qPCR with 4 different RNA...

20 June 2018 3,637 1 View

What is the difference between delta CT and delta delta CT in RT-qPCR?

These 2 analyze methods are recommended for specific gene expression analysis?

01 June 2018 6,559 4 View

What methodology is more accurate to measure mRNA amount: Qubit or bioanalyzer?

I have analyzed mRNA human samples in bioanalyzer and the RNA concentration provided did not match with the RNA concentration of the same sample in the same dilution provided by Qubit. What RNA...

04 May 2018 2,833 3 View

How much sythetized pure oligo can I use for a standard curve in qPCR?

I am trying to define a standard curve for the primers that I have designed for qPCR and I ordered to sythetize the correpondent oligo for test the primers in different concentrations, but I don't...

29 November 2017 5,347 2 View

What's the best qPCR kit to detect cDNA from low RNA amount?

I have gotten a low RNA amount from human dendritic cells using RNeasy kit from Qiagen, such as 11ng/ul and I need to perform a qPCR assay from these samples. Or trizol still is the better way to...

22 February 2017 989 3 View

What's the best method to isolation RNA from animal cells in culture?

I have tried to isolation RNA from human dendritic cells using RNeasy kit (QIAGEN), but I got a very low yield of RNA, such 30ng/ul. In the last time, I tried to combine QIAGEN protocol with...

19 January 2017 8,386 3 View

What's cytokines that dendritic cells are able to produce?

I would like to know what cytokines are produced by dendritic cells. I need to know ,specifically: These cells can produce IL-1beta, IL-6, IL-10, IL-12p40, TGF-beta, and TNF-alfa?

15 January 2017 3,184 3 View

How is the best way to obtain a good yield of metacyclic trypomastigotes from a epimastigote culture of Trypanosoma cruzi?

In the lab where I work, it's used the experimental protocol of Canavaci et al (2010) in order to elicit metacylogenesis in epimastigotes from CL Brener strain cultivated in LIT medium. Then,...

11 January 2017 3,216 2 View

What is the most recommended method to determine the best amount of antibody to detect a specific protein in flow cytometry?

I have performed a antibody titration to detect a specific cytoplasmatic protein in human monocytes starting with the double amount of general antibody recommendation (2 ug) and I have made 9...

01 January 1970 693 1 View

What is the difference between separation index and staining index in antibody titatrion to flow cytometry?

I have observed that both index are used in order to evaluate the best concentration of antibody in FC, but which one is most recommended to discover the best antibody concentration that provide...

01 January 1970 2,820 4 View

Where I can purchase validated and reliable siRNAs to silence a target gene?

I intend to silence a gene in human monocytes-derived DCs or THP-1 cells using RNAi to study its role in a infection. Where I can purchase validated and reliable siRNAs to silence a target gene?

01 January 1970 8,994 2 View

Why we can not use serum on cell culture media for protein precipitation and western bloting/immunobloting analysis?

I have found 2 protocols for caspase 1 detection from cells supernatant and both said to avoid fetal calf serum (FSC) or other kinds of serum to the media before the stimulus and to collect the...

01 January 1970 4,757 2 View