How to transfect hippocampal neurons at DIV2 without the cells clumping together or fasciculating?

Saakshi Champaneria @Saakshi-Champaneria

30 April 2024 0 8K Report

I am working with primary hippocampal cells from E18 rats. The problem I am facing is with the transfection of these neurons in a 12-well plate with Lipofectamine 2000. When I transfect at DIV 2 or DIV 5, the cells start clumping together and fasciculating. I tried repeating it multiple times but facing the same issue. Can someone help me with this?

Badges
Science topic

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

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 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

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

What are the pros and cons for mice versus rats?

Hi Everyone, I am designing a pilot study using rodents to assess the effects of simulated space environments (radiation + altered gravity) on microbiome and immune system. I have limited...

24 February 2021 1,825 3 View

How can I stain the somatodendritic part of inhibitory neurons in a hippocampal culture with high specificity?

I would like to distinguish the somatodendritic part of the inhibitory neurons in a hippocampal culture (where we expect to have 5-10% of GABAergic neurons). What are the best antibodies and...

23 February 2021 6,756 1 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