Which transfection reagent is best with reporter assays on MIN6 cells?

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What is the meaning of pale stained nuclei with hematoxylin?

I am staining pancreas with HE. I found beta cells show pale hematoxylin staining in conditional knockout mice compared to surrounding other islet cells, exocrine and acinar tissue " this means...

03 April 2018 363 1 View

Normalization by DNA content in glucose stimulated insulin secretion assay for islets?

I want to ask about normalization step: if I want to normalize by DNA content how I should isolate DNA after treatment of the islets with acidified ethanol?

07 August 2017 4,491 0 View

Has anybody faced difficulties with islets isolation from db/db mouse model?

Recently, I am trying to isolate islets from db/db mice. I use the same protocol I regularly use for islets isolation from mice but I can't get any islets or very few pale islets attached to...

01 February 2017 5,966 7 View

For murine islets, can I work on islets extracted from one mouse or I have to pool from several mice?

I am recently extracting murine islets. I see most researchers recommend pooling islets from several mice. I just want to check expression of my protein of interest in islets as an initial stage...

10 November 2016 9,415 3 View

What is the best internal control for western blots of mice lysates?

I have been using tubulin as internal control for my western blotting analysis of mice islet lysates. I wonder if there is a better internal control the researchers working with mice islets use.

09 October 2016 6,299 3 View

I am asking if somebody previously transfected islets of Langerhans isolated from mice with siRNA before. Is this going to work?

If this works what kind of tranfection reagent is recommended for the islets?

07 August 2016 4,533 9 View

Can I treat mice islets expressing tamoxifen induced cre in vitro with tamoxifen to knock out a floxed gene?

I have a mouse model that expresses modified Cre recombinase, so that only upon tamoxifen treatment of the animals they express Cre, thus KO of floxed gene can be induced. I want to isolate the...

07 August 2016 2,715 2 View

Are beta actin and tubulin levels in islets affected by fasting ?

I am interested in knowing the expression of a certain protein in the islets under feeding and fasting conditions. I use the same total protein concentration for immunolotting. In my first trial I...

07 August 2016 4,351 1 View

I want to ask about measurement of insulin content in MIN6 cells?

I did a glucose stimulated insulin secretion assay for MIN6 cells. I measured the released insulin in the Kreb's Ringer buffer containing certain concentrations of glucose. In the end I collected...

06 July 2016 3,929 2 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

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

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 my colony PCR results of my recombinant bacterial not showing any results?

I am performing ligation of the plasmid and a target gene. The steps I have taken are: 1. Double digestion of the plasmid and target gene 2. Ligation of the plasmid with the target gene 3....

05 August 2024 2,570 3 View

How to isolate lymphocytes from mouse spleen?

I have tried several times to isolate lymphocytes from mouse spleen, but all attempts have been unsuccessful. I tried most available protocols. I used different dissociation media (HBSS with Ca...

04 August 2024 9,913 7 View

To perform transfection with DharmaFECT Duo in AGS cells. Could you tell me what the ideal concentration is to avoid significant cytotoxicity?

I would like to perform transfection with the reagent DharmaFECT Duo (Horizon) on the AGS cell line. Could you please inform me of the optimal concentration to use without causing cytotoxicity in...

03 August 2024 3,851 1 View

Transfection in HEK293T cells?

Dear All, I am trying to transfect a pCDNA3.1 vector containing my gene of interest. The purpose is to figure out the localization of the protein of interest. I have fused the protein with GFP on...

31 July 2024 9,892 4 View

What Are the Best Alternatives to the MTT Assay for Assessing Cell Toxicity of Colored Herbal Extracts?

I am currently investigating the cytotoxicity of a series of herbal extracts, and like many studies, I have been using the MTT assay to evaluate cell viability. However, I am encountering a...

31 July 2024 193 4 View