Sharon Lee

35 Questions 10 Answers 0 Followers

Questions related from Sharon Lee

Does company brand of cytokines affect differentiation and/or (M0/M1/M2a) polarization of TH1-P cell line into macrophages?

Concerned that there will be functional differences between products from Peprotech vs. Mitenyi vs. R&D.

06 June 2018 7,342 0 View

What does it mean when transfected macrophages increase in size (forward scatter) and granularity (side scatter)?

Cells treated only the transfection agent or the transfection agent with the miRNA construct (meant for transfection), had a higher forward scatter and side scatter compared to cells that were...

05 May 2018 1,119 1 View

Any suggestions on how else I can label the nuclei of a cancer cell line that has formed a 3D cellular aggregate?

I used NucBlue Live from ThermoFisher (https://www.thermofisher.com/sg/en/home/references/protocols/cell-and-tissue-analysis/protocols/nucblue-live-readyprobes-protocol.html). I found that this...

05 May 2018 3,834 1 View

Any suggested tricks or protocols to lift off primary human macrophages from well plates without scraping?

And does scraping activate cells?/ damage surface proteins on cells?

05 May 2018 4,320 0 View

Any comments on whether median fluorescence intensity (MFI) or geometric mean is better to evaluate CD marker expression using flow cytometry?

. .

05 May 2018 6,484 0 View

Any suggestions on a non-toxic nuclear dye for labelling macrophages while the cells are live?

NucBlue LiveReadyProbes by ThermoFisher (https://www.thermofisher.com/sg/en/home/references/protocols/cell-and-tissue-analysis/protocols/nucblue-live-readyprobes-protocol.html) seemed to seriously...

05 May 2018 5,783 3 View

Is it neccessary to use filtered pipette tips when adding lysis solution to lyse cells for downstream RNA work?

Or is the use of filtered tips only crucial from the point of isolating RNA from the lysate? (i.e. after obtaining the lysate)

04 April 2018 9,424 5 View

Any suggestions on how I can inhibit secretion of cytokines by a cancer cell line without killing the cells?

These 'secretion-inhibited' cancer cells would then be co-cultured together with myeloid cells so I am hoping the environment would not be toxic to both cell types.

04 April 2018 5,149 9 View

Can anyone share how they label monocytes with CellTracker Orange(CMRA)?

I use IMDM as staining buffer (is this ok?),but lose 60% cells after staining. Also, any idea/comment on why we should avoid amine-containing or thiol-containing buffers as the staining buffer?

12 December 2017 2,417 0 View

What markers can I use to characterize macrophage subsets (M1 and M2)? And what about different monocyte subsets (e.g. patrolling, etc)?

For immunostaining of cells that are fixed.

07 July 2017 7,517 3 View

Comments on the easiest method to attach antibodies to paper for dry storage, where antibodies can be rehydrated when it is required for use?

06 June 2017 5,416 0 View

Comments on the simplest method to conjugate gold nanoparticles to antibodies?

06 June 2017 7,090 0 View

Will I kill HUVECs when I spin/pellet them down at 700G, 4degrees?

spun in v bottom multiwell plates

06 June 2017 2,626 0 View

How can I retrieve Tcells from a collagen gel matrix without harming cell viability or altering their expression of surface markers (i.e. phenotype)?

I intend to culture cells in a 3D matrix, and retrieve cells at certain timepoints for downstream phenotyping (by flow cytometry). I heard that the process of retrieving cells from a gel matrix...

06 June 2017 1,924 0 View

Comments on the simplest/most convenient method to isolate autologous HUVECs from humans?Comments on how easy/dificult it is to culture them in vitro?

Wondering how long these cells can survive in culture, and if cells have special requirements.

06 June 2017 5,815 1 View

What happens at the molecular level when HLA-mismatched monocytes and T cells (mostly CD8+) are co-cultured in vitro? Is there allogenic reaction?

Would T cells kill monocytes? Would monocytes stimulate/activate T cells?

04 April 2017 7,373 6 View

Can we compare MFI values if a different panel (i.e. set of colours), machine is used? Also, would compensation alter the comparison?

Cells cultured in the same conditions and stained using the same procedure.

03 March 2017 8,596 0 View

Any tricks to lift off monocytes without the use of Trypsin?

Important that surface markers are not "chopped off" as cells are used in flow cytometry

02 February 2017 4,995 3 View

Would I expect to see lowered CD14+ expression from a population of total (unsorted) monocytes that are dying?

Cells in culture for 72h.

01 January 2017 8,261 1 View

If I want to compare MFI values across different experiments, is it important that I use exactly the same PMT voltages? Is slight differences be ok?

Slight differences meaning PMT voltage of 450 instead of 420. (just an example)

01 January 2017 4,962 8 View

Any comment on the stability of IL-2(human) if it is added as a supplement to cell culture media, and stored in 4deg, in the dark for 1 week?

Would there by any difference in cell activity/functionality if they are cultured using the above-described media vs media that was added with IL-2 at the point of culture?

01 January 2017 1,172 0 View

What is the effect of milliQ H20 on cells if cells are only exposed to this for a few seconds?

I accidentally spilled some sterile milliQ water on my cells that were already in culture media. I immediately removed all the media and replaced it with fresh media. In my case, sholud I be...

12 December 2016 2,108 6 View

Is it common for intracellular cytokines such as IFNg to be detected at much lower levels than postitive cntrls (PMA/Ionomycin) or other donors?

I'm uncertain if it's a technical issue that I need to troubleshoot and resolve.

12 December 2016 7,545 6 View

Any issues that would arise if I used 5X higher the concentration of live/dead stain?

I am thinking it would not matter for my analysis since live cells would not be stained with the live/dead stainining reagent....am I wrong?

12 December 2016 6,190 1 View

What is the longest that T cells can stay alive on ice?(Important for transporting cells between venues). What can I do to protect their health?

Is 4 hours too long?

12 December 2016 8,273 3 View

Incubator door was left ajar for probably 30min. Should I be worried about changes in behaviour/function of the cells that were being cultured there?

Observed some evaporation of the culture media...but inside of incubator felt mostly warm (i.e. 37deg).

12 December 2016 3,384 2 View

Any advice on how to speed up collecting/lifting off cells from well-plate, while protecting cell viability & cell presentation of surface antigens?

Cell types: HepG2, Monocytes, T cells Note: Collected cells will be stained for surface antigens, and flow cytometry is used to measure for expression levels.

11 November 2016 7,302 3 View

Will concentrating supernatant (via Amicon filters) give rise to inccurate/biased results in luminex measurements?

Especially in the case where supernatant may be low in concentration, whereby supernatant was derived from a low density of cells in culture.

11 November 2016 1,971 1 View

Can someone advise on what I could do to increase the sensitivity of an ELISA or luminex assay for cytokines in co-culture supernatant?

Previously, we could not detect any cytokines. We suspect that supernatant is dilute due to few cells in culture. (Increasing cell density is not an option for us.

11 November 2016 4,084 5 View

For cell culture of HepG2, is there difference in using RPMI (already with L-glutamine) vs. RPMI (L-glutamine only added at the time of cell culture)?

Heard some researchers have always purchased RPMI (no L-glutamine), and only added L-glutamine when preparing the complete cell culture media. Wondering if this has to do with the...

11 November 2016 5,115 3 View

How can we detect for conjugated proteins on a cell surface?

We suspect that the 'ferocious' immune response that we observe is due to the presence of a conjugated ligand.

11 November 2016 6,500 3 View

How long can cells stay on ice (cells collected from well-plates with PBS/EDTA) without significant modulation/internalization of surface antigens?

Read that temperature can cause modulation and internalization of surface antigens and this can affect fluorescent intensity in flow cytometry measurements.

11 November 2016 4,146 7 View

Are there any issues with staining cells with an antibody concentration that is 1.2X the concentration recommended by the manufacturer?

Antibody used to stain for expression of inducible surface protein.This is a primary conjugated antibody for flow cytometry. Fluorophore: Brilliant Violet 711 (Similar to Qdot711); Considered to...

11 November 2016 3,826 4 View

Am I reducing the concentration of cytokines (i.e. IL-2) when I filter through a 0.2uM pore size filter?

IL-2 is provided in powder form. To prepare, I dilute in millipore water and make small aliquots. When preparing cell culture media, I add the IL-2 stock to the media. Then, I filter the...

11 November 2016 8,750 2 View

Any input on the use of serum-free media to culture monocytes or HepG2 cell line for (only) 24h (as opposed to using media with 2% human serum)?

Particularly, the media is AIM-V (see link) by Gibco, which the company claims can be used without serum to culture certain cell types....

10 October 2016 6,813 1 View