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

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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....

11 December 2019 3,903 3 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...

04 May 2019 3,583 4 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...

04 May 2019 5,123 5 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...

04 May 2019 5,766 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...

04 May 2019 7,741 2 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...

04 May 2019 1,552 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...

04 May 2019 10,433 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...

04 May 2019 6,758 4 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

31 December 2018 5,290 5 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

10 November 2018 6,793 5 View

Why Do TDS and EC Increase with Larger Wastewater Volumes, While BOD and COD Decrease?

I have carried out MFC experiments on three different volumes, 50, 500 and 1000 mL of wastewater. Results after MFC treatment shows that TDS and EC are more in larger volumes of water i.e. TDS and...

09 August 2024 9,621 0 View

Could it be a cell culture contamination?

Hello everyone! I observed in my culture (htert-RPE1 cells) an orange- red particle at the bottom of the dish. It is visible to the naked eye as a very very small red dot. Could it be a...

09 August 2024 2,824 3 View

How to confirm the site-directed mutagenesis result without performing NGS?

I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...

08 August 2024 4,645 2 View

Is there any way to quantify bacterial and fungal cells in their mixed culture?

I am working in fungal fermentation of soybean meal and there is bacterial growth in them at times. I am trying to quantify fungal cell counts and bacterial cells; but I haven't been able to do at...

07 August 2024 7,535 4 View

How to normalize and take the significance of the MTT OD values with 3 replicates for the same cell-line?

Hi, I have a question about normalizing the MTT OD values for doing the statistical analysis. So, if we have 3 different plates and we call them 3 different replicates, so, first we would...

07 August 2024 8,106 4 View

Why activated CAR-Jurkat cell could not kill targets?

Previously when I co-coluture anti-CD19(FMC63) CAR-Jurkat with Raji with E:T=5:1, Jurkat can eliminate Raji in 24h. However, when I test another CAR construct, although I can dectect totally CD69...

06 August 2024 641 2 View

Weak DAPI staining after immunohistochemistry - how to improve?

After immunohistochemistry of previously fixed in PFA and EtOH and then frozen 20 μm sections of zebrafish brain, DAPI staining is very weak (right) compared to the same sections stained without...

05 August 2024 9,637 2 View

Where should the ARS sequence be introduced on a plasmid?

Hi all, I need to introduce an ARS (autonomously replicating sequence) in my plasmid but I'm not sure which position would be the best. Does anyone have any suggestion? A picture of the plasmid...

05 August 2024 1,573 4 View

Why after performing site directed mutagenesis ,I don't see any colony after transformation?

I want to introduce a point mutation (change in one nucleotide) into my gene of interest (DNA binding domain) I have designed primers as recommended on the Data sheet of the kit : -Both primers...

05 August 2024 9,059 3 View

Why did the authors extrapolate a phenotype that they experimentally proved in one bacterial strain across the whole genus of the organism?

I aim to be as skeptical as possible regarding whether a pair of orthologous genes results in the same phenotype in their different but related bacterial organisms under similar environmental...

05 August 2024 6,787 4 View