Does anyone sharpen their surgical scissors in the lab? If so, what method do you use?

Hi all,

I am wondering if anyone has any experience with using InvivoGen's Primocin antibiotic solution beyond the indicated expiry date when properly aliquoted and stored at -20degC. We have a relatively old stock in the lab (more than 1 year past the expiry date) and wondered if they are still effective to supplement culture media for maintaining primary human cells.

Alternatively, could anyone suggest simple tests to help determine the effectiveness of the antimicrobial activity of our 'expired' antibiotic solution?

Many thanks for your help and suggestions, Randy

Please i have been trying to dissolve Palmitic acid in BSA-NaOH, it keeps solidifying after heating. I wanted to used DMSO but its' lethal to cells. And also the DMSO recommended concentration to cell/medium should not exceed the range 0.1-0.5, and this concentration couldn't dissolve it.

PLEASE GUIDE ME THROUGH.

Thank you.

I have multiple sets of RNA-seq data and I want to compare gene expression between control and treated groups. My interest extends beyond differentially expressed genes; I also want to identify non-differentially expressed genes. I understand that Log2FoldChange and p-adj are commonly used to define differentially expressed genes. Alternatively, genes that fail to meet the criteria for differential expression are considered non-differentially expressed.

However, classifying a gene as non-differentially expressed does not definitively indicate that the RNA-seq data confidently establishes the absence of changes in gene expression. For instance, this could be attributed to substantial within-group variation or low gene counts that hinder unambiguous determination of expression levels. So, how can I effectively distinguish truly non-differentially expressed genes from those exhibiting significant within-group variation or yielding very low counts? Are there any software packages available for this purpose? Alternatively, are there established statistical methods or standards that can guide me in this regard?

I am seeing a gene expression through Rtpcr .so wanted to know which method I should use for gene expression .is 2^-delta ct method I can use over 2^-delta delta ct

Hello! We have a rather insoluble drug in aqueous buffers but it gets dissolved in DMSO. It also remains soluble after we add 95% of PEG300. Would 5% DMSO/95% PEG300 would be acceptable for oral gavaging mice every day for 30 days approximately?. Unfortunately, if we add water or saline, the drug comes out of solution. I would really appreciate it if anybody has any input on the matter. Thanks!!

Hi all,

We filtered seawater and preserved the filters in RNAlater on site. The tubes with the sample in the RNAlater were placed in a cold box until arrival in the lab where they were left overnight at 4C. After this they were placed in a freezer at -20C still in the RNAlater until they were processed a couple of weeks later. When we retrieved the samples for processing we realised that some of the samples where frozen while others weren't, there doesn't seem to be a reason for this difference (other than maybe some samples where a bit more wet than others?).

My questions are:

1. Is this a problem in the long term? There seems to be a bit less RNA in the frozen samples (measured with the nanodrop) but very minor and we did process the samples soon after arrival in the lab, will this be a problem if we store the samples for long periods of time?

2. Is there a way to avoid this? Was this caused by extra moisture in the sample?

Maybe I am looking this the wrong way around and samples are supposed to freeze and they didn't because the salt content or another reason and we should aim at freezing them.

Any thoughts will be greatly appreciated.

Hi all,

I'm running seahorse assay on primary hepatocytes to measure mitochondria activity. I always plate each well with same cell number. But they could still be different after 2-3 days of culturing. And it makes no sense for comparison if the cell numbers vary significantly among wells. So I tried two different ways to normalize the cell number:

a. Measuring protein content using BCA assay.

b. Measuring DNA content using Picogreen staining.

However, these two assays gave me different result. I'm wondering:

1. Which assay is better for cell number normalization?

2. Does BCA assay or Picogreen staining also take into account dead cells? If they both do, which one is more accurate in measuring live cells?

Would be great if anyone who had experience could give me some suggestion. Many thanks.

Best,

Surui

I would like to try activating B cells (or B cell lymphomas) by adding an anti -IgM antibody. Does anyone have experience in doing so? Should I rather use a whole antibody or (the more expensive) F(ab)2 fragments? Which concentration should I try for a start?

Thank you!

Anna

Hello everybody,

I immunized mice successfully with NP-KLH ( 4-Hydroxy-3-nitrophenylacetyl hapten is conjugated to KLH (Keyhole Limpet Hemocyanin) lysine through amide bonds). I let them sit for four weeks and took a blood sample each week. Now I want to do an ELISA for checking how many high and low affinity antibodies (i.e. NP-specific IgG1) there are in each sample comparing different genotypes. Technically the ELISA is ok, but I attached a protocol anyways.

The problem is that we are running out of standard for NP and the TA who did this is not here anymore and also didn't leave a protocol. With the old standard (where I don't know how it was made, how long the mouse was immunized etc) everything is fine, I see more binding on the NP27 coated site of the plate. But with simply using serum from previously immunized mice as a standard it does not work. I attached an example of this where I used the sera of the same mice and on the plate with St NP (= old standard) everything is fine, but with St Tea (new standard) it does not work.

When I look into the literature the only description I find is that you use serum from previously immunized mice, but that doesn't seem to work. So I think, maybe I need to immunize the multiple times or longer or dilute the standard in some way?

Could anyone please share with me a protocol for making a new standard for NP ELISAs? For other standards you can simply buy them, i.e. IgG standard, but with NP I didn't find anything similar.

Thank you so much for your help in advance!

Best,

Laura