Amanda Rocha

38 Questions 12 Answers 0 Followers

Questions related from Amanda Rocha

Could I trust in qubit for protein quantification?

I need quantify the protein amout from human DCs lysed by RIPA buffer ( 150 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0 ) to use in a immunoblot....

12 December 2019 3,848 3 View

What is antibody concentration recommended to western blot?

I need to detect and measure the abundance of cytoplasmatic proteins (CASP1 an GSDMD) by western blot in human mo-DCs, and some companies where I purchased the antibodies do not specify a...

05 May 2019 1,485 5 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...

05 May 2019 10,179 3 View

Is it possible to use the same permeabilizing agent to perform intracellular staining of cytoplasmatic and nuclear proteins in flow cytometry?

I work with human mo-DCs and I need to detect cytoplasmatic and nuclear proteins by flow cytometry using flurochrome-conjugated antibodies (direct detection), that is, I need to permeabilize both...

05 May 2019 5,067 5 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...

05 May 2019 3,506 4 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...

05 May 2019 6,679 4 View

There is a need to fixation step to detect soluble proteins in cytoplasma or nucleus by flow cytometry?

I need to detect and measure the abundance of cytoplasmatic proteins (AIM2, GBP1, GBP4) and nucleic proteins (IFI16), all apparently soluble, using direct detection by flow cytometry with...

05 May 2019 5,712 3 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...

05 May 2019 7,670 2 View

How many human primary cells I need to quantify protein abundace in western blot analysis?

Someone that work with human primary cells and western blot: how many cells I need to identify protein abundance in western blot analysis? I want to quantify Caspase-1 and gasdermin D

01 January 2019 5,221 5 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 1,299 3 View

Could I differentiate human dendritic cells from cell line? Which one?

Someone have ever tried to differentiate human DCs from cell line that resemble mo-DCs?

11 November 2018 6,326 5 View

How I can detect caspase-1 activation in human monocytes-derived dendritic cells in culture?

IL-1B/IL-18 secretion, westen blot analysis of 2 subunits of caspase-1, FAM-FLICA detection or another method?

11 November 2018 2,536 2 View

What is the most efficient method to transfect human cells to knockout mediated by CRISPR/Cas9?

Or it depend on the kind of plasmid used?

11 November 2018 994 2 View

What I can use as caspase-1 activator?

I need a positive control to my caspase-1 activation assays in human monocytes-derived dendritic cells in culture.

11 November 2018 7,557 3 View

Where do you recommend to purchase human antibodies to proteins to Western blot and flow cytometry?

What is the best brand of antibodies to detect human proteins through WB and/or flow cytometry?

11 November 2018 452 4 View

Which is the best way to compare a intracellular protein level between control and treated group: western blot, flow cytometry or another one?

I need to analyze a specific intracellular protein level in control and infected human monocyte-derived dendritic cells

11 November 2018 6,741 5 View

Can I trust in FAM FLICA™ Caspase 1 Assay Kit to determine caspase-1 activation?

I want to detect caspase 1 activation in human monocytes-derived dendritic cells after interaction with my pathogen.

11 November 2018 2,183 4 View

How to detect pyroptosis?

How can I detect pyroptosis in human mo-DCs induced by a pathogen? Are there some comercial kit?

11 November 2018 5,206 0 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...

05 May 2018 7,664 3 View

Can I perform qPCR with a multichanel pipette?

Someone already tried to perform qPCR with a multichannel pipette? Or is it not recommended?

05 May 2018 7,232 6 View

Is there any company that sells primers to human genes already validated?

I need to perform qPCR of 40 human genes and I would like to know if I can purchase primers that already have tested and have confirmed efficiency of >90-95%

03 March 2018 4,459 4 View

There are difference between 25uL and 50uL of final volume in qPCR?

I’ve been performing, so far, my qPCRs reactions in 50 uL of final volume; however, at doing in this way, I have spent a lot of material. In this sense, I would like to know if I could perform my...

03 March 2018 9,164 4 View

If I lower the final volume of the qPCR reaction from 50 uL to 25 uL, will it interfere in the analysis of my replicates?

If I switched the final volume of my qPCR reactions from 50uL to 25uL, will it interfere in my analyzes of my biological replicates, once I already started the analyzes of some replicates with 50...

03 March 2018 699 4 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...

11 November 2017 7,950 2 View

RNA is stable in high or low temperatures?

How much I can rise or decrease temperature without compromise RNA integrity?

08 August 2017 2,345 3 View

How can I inactivate DNAse from my RNA samples?

DNAse protocol that I am using in my RNA samples says to inactivate DNAse using phenol-cloroform and shows a complex and prolonged protocol to inactivate. There is an alternative method that I can...

08 August 2017 9,165 3 View

How bioanalizer can determine the RNA integrity?

What's the method used to measure the RNA integrity?

05 May 2017 8,215 2 View

Why A 260/280 to measure RNA amount?

Does anyone can explain why I need to use A 260/280 to measure RNA amount? What this means?

05 May 2017 6,789 4 View

How is determine the RNA integrity number (RIN)?

How bioanalyzer determine the RIN of RNA sample? Why in some cases the RIN appears as not applicable? What could happened?

05 May 2017 3,373 4 View

How can I differentiate monocytes into dendritic cells?

How method I can use to obtain dendritic cells from monocytes? Or there is a better way to obtain high amount of these cells?

01 January 2017 508 2 View

What's the difference between ELISA Quantikine® kit and Duoset ELISA kits?

What's the major difference about using these two kits for quantify cytokines in supernatant of culture cells? Both have the same aims?

01 January 2017 5,930 2 View

How can I harvest and storage supernatant from cultured cells and perform a ELISA assay for cytokines present in this supernatant?

I have co-cultured dendritic cells with T. cruzi and I need to evaluate the cytokines realized from this interaction. How can I harvest and storage the supernatant after the infection to perform...

01 January 2017 3,418 12 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?

01 January 2017 2,345 4 View

How is the best ELISA kit to quantify cytokines from the supernatant of cultivated cells in vitro?

I need do quantify the cytokines that are produced from dendritic cells descendant from monocytes when I infect these DCs with Trypanosoma cruzi in a 24-well-plate, but I have never worked with...

12 December 2016 8,038 11 View

Which is the best way to transfect human dendritic cells?

Could someone that work with human mo-DCs transfection with siRNAs recommend a best method to do this? I generally have seen works using lipofectamine 2000 (Invitrogen), eletroporation (Amaxa/bio...

01 January 1970 3,312 3 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 1,630 0 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 3,954 4 View

How many human dendritic cells I need to identify protein abundance in western blot analysis?

Someone that work with human dendritic cells and western blot: how many cells I need to identify protein abundance in western blot analysis? I want to quantify Caspase-1 and gasdermin D

01 January 1970 2,513 4 View