Is it okay to use ciprofloxacin in cell culture media as an antibiotic?

Hi y'all,

I am running into some difficulties with qPCR (SYBR). To give y'all a brief background summary, I transfected some cells (ASO) and am running qPCR to see its effect on known genes using qRT-PCR. The first time I ran a qPCR, I saw little to no amplification. I re-ran it again and this time I went back to the beginning (RT-PCR from the original RNA) and was able to get some data. However, I am running another qRT-PCR but this time it's not working at all, as in I am seeing zero amplification, which is weird because, again, we know for a fact that some of the genes are supposed to show up in abundance. I was very careful throughout the whole process, so I don't think there was any human error. I haven't run a gel yet, so that's something that I have in my mind. I just wanted to see if y'all have faced similar problems.

Thanks in advance!

I was always told that after lysing the cells, spin them, and only take the supernatant or storage, but why? why do we need to spin the cell lysis to remove the pellet?

Hi everyone,

I'm looking for adding Amphotericin B (Fungizone) to my cell culture, as I'm frequently see contamination, specially, fungal contamination in both flasks and plates. (I'm working with neuronal cell culture (SH-SY5Y).

Has anyone working with Amphotericin B (Fungizone) for cell culture before?

I saw Amphotericin B (Fungizone) in both Gibco and Sigma, which one is better to use?

Thank you.

I accidently added five fold more chloroform in the initial steps of RNA isolation. What are the consequences of this? Also, if i add more isopropanol to compensate this effect, how both chloroform and isopropanol are related ?

I am using THP-1 cells and i want to remove the dead cells from the cellular suspension.

Hi all,

I am recently doing my research in macrophage polarization with Raw 264.7 and be refused by the experimental dates.With FBS it showed really low expression in both CD86 and CD206 whether adding LPS or IL-4.As consequense we tried heat inactivated FBS and the expression surpassed 70.However,when using heat inactivated FBS,CD206 didn't go up with IL-4 while went up significantly with LPS.Is the problem from heat inactiveted FBS(it is too strong?) if it is what can i do? change the inactivating way?Or there was something else to cause the problem?

Thanks for any advice.

For culturing i used RPMI 1640 media and 10%FBS(whether heat inactive).To polarizate macrophage i used LPS(10ng/ml 48h) and IL-4(100ng/ml 48h) for M1 and M2 respectively.For detaching i used trypsin and PBS.

Why is the Western-blot band connected into a line? As shown in the picture, it's the mouse GAPDH band and it should be 10 bands separately in the gel where the marker is on both sides.

Hi everyone,

we just recently started working with THP-1 cells. We aim to use THP-1-derived macrophages as a model system to assess the effect of transient knock-down of our target gene on their cytoskeleton.

The differentiation protocol we use is 72h of treatment with 30 nM PMA, followed by a resting time in PMA-free media for another 72h. After this time, we perform the knock-down and wait again 72h before doing functional experiments.

The problem now is, that on the day of functional experiments, the cell appear to me a lot more like dendritic cells that macrophages, i.e. they have a massive amount of protrusions. In the literature, I only find protocols adding additional factors (IL-4, TNFa, GM-CSF) to generate dendritic cells...

Does anyone have experience with 'accidential' dendritic cell differentiation in THP-1 cells? What could we be doing wrong? Is the time between PMA treatment and functional experiments too long?

As we just started recently with this, we have very limted experience with THP-1, and any comments and suggestions (even the most basic) will be very much appreciated.

Thanks in advance!

Best,

Ramona

what is most frequently used concentration of sodium azide to kill microbial culture?