How to induced apoptosis/Necrosis to get high% for PI positive group in CFSE/PI mediated CAR-T cytotoxicty assay ?

i wanna perform CAR T cell mediated cytotoxicity assay by using CFSC/PI. As PI cannot cross the membrane of live cells, making it useful to differentiate necrotic, apoptotic and healthy cells. i...

11 December 2018 4,273 7 View

How to Calculate the concentration of DNA plasmid from stock for qPCR ?

Hey all, I get puzzle in a simple calculation. I have a protocol for qPCR, according to instruction i need 5ul of DNA having the concentration of 1ng/ul, for starting a standard curve. My DNA...

09 October 2018 9,738 3 View

How to calculate molecular weight of protien from a sequence ?

Hey All, I have a peptide composed of various domains. I have the sequence of whole peptide and individual domain. i wanna express that whole peptide on T cell. After expression, i wanna run the...

07 August 2018 1,896 12 View

Cell line to target CD4 and CCR5 ?

I design some cellular therapy to target HIV, my cellular therapy has the potential to eradicate CD4 cells and inhibits and the biding of CCR5 receptor during the entry of HIV genome in host cell....

06 July 2018 5,145 0 View

How to store Blood plasma ?

I collect blood from hPBMC mice and centrifuge it to get the RBC and plasma. I need to store the plasma for future experiments like Cytokines resale studies and other. what is the best way way...

04 May 2018 1,177 0 View

How to stain and fix cell for flow cytometry for long time ??

I infected the 293tt cell with AAV virus to check the expression of my CAR molecule. According to protocols, I will collect the cells and wash in PBS, than i will stain with a antibody (Fab...

03 April 2018 2,836 5 View

How to calculate the Genome copy number of AAV titer by qPCR ?

I run qPCR to titer the AAV ( which i got by transfection in 100mm dish). I make four dilution of my sample ( 12 well and triplicate). I take GFP plasmid as a standard and were diluted to 1ng/ul,...

03 April 2018 8,447 2 View

Transfection and Co-transfection ?

Hello everyone, I am beginner in molecular biology. I am confused with the term transfection and co-tranfection. Can someone explain me what is the basic difference between them ? i search...

02 March 2018 5,654 0 View

How to determine the optimum voltage and time for a better separation in gel electrophoresis and westron blotting ?

02 March 2018 4,460 5 View

How to prepare AAV viral stock after transfection?

I am going to transfect AAV293 cell with the AAV plasmid by adopting the PEI protocol. I wanna ask what is the best way to collect the viral stock ? In online literature and methods there are...

01 February 2018 3,922 4 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 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

Does RNAiMAX transfection reagent work in serum free medium?

I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium. Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in...

26 February 2021 10,041 3 View

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View

How do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?

I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...

25 February 2021 6,635 5 View

What is a good reagent for lentiviral plasmid transfection in HeLa cells?

I have been using Lipofectamine 2000 but was hoping there may be a better protocol or one for something like Lipofectamine PLUS reagent.

24 February 2021 1,633 4 View

How to use protein A G beads to purify antibody?

Secretory universal influenza antibody CR9114 were successfully expressed by adenovirus vector ,then we used protein A G beads purify CR9114.But the molecular weight of the purified antibody under...

22 February 2021 4,318 2 View

Why the cells die after stable transfection?

I tried stable transfection in the U87 cell line with the sgRNA-Cas9 All-in-One lentivector. Cells were cultured in a 6-well plate, 4ug plasmid was transfected after 24hs when the cell confluence...

22 February 2021 2,864 4 View

Is there a method for evaluating nanoparticle adhesion to epithelial tissue in vitro?

I want to measure how well nanoparticles can transfect cells based on NP adhesion properties. In the literature I find people do it in animal studies, and epithelial adhesion helps in transfection...

21 February 2021 7,791 3 View