Sharon Lee

19 Questions 6 Answers 0 Followers

Questions related from Sharon Lee

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...

07 May 2018 1,437 1 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.

24 April 2018 7,792 8 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?

19 December 2017 4,022 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.

05 June 2017 3,787 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?

06 April 2017 6,484 5 View

How to compare MFI values/Geometric mean from analysis using different software?

I am trying to figure out the equivalent MFI (Median fluorescence Intensity) for analysis done using Flowjo and analysis done using Kaluza. Any help would be much appreciated. Thanks.

24 March 2017 9,249 4 View

To compare MFI of protein X across experimnts,can I change PMT voltage for identifying markers(e.g. CD8) while PMT voltage for Protein X is unchanged?

I am wondering if the MFI value is only dependent on the PMT voltage of the corresponding laser, or if the compensation matrix/ entire panel (and associated set of PMT voltages) affects the final...

26 January 2017 1,773 1 View

How long can I leave fluorescently-stained cells in staining buffer (FACS buffer) on ice so that measurements are not altered (Cells are unfixed)?

Is 4 hours too long? Would the MFI signal decrease by more than 50% of the expected value?

18 January 2017 3,165 4 View

Any issues with centrifuging cells at 800G, 4deg, 3min without first breaking ug the pellet?

After centrifuging in a v-bottom 96-well, at 2000rpm (800G), 3min, 4deg, (volume about 180ul) I added 180ul buffer without breaking up the pellet, and then centrifuged the cells again. (i.e i...

18 January 2017 989 1 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).

14 December 2016 2,163 2 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...

13 December 2016 5,762 5 View

Any suggestions on how I can upregulate PD-1 on CD8 T cells in the shortest period of time possible?

Population is majority CD8 T cells (but not 100% pure CD8 T cells)

05 December 2016 9,532 1 View

What could have caused the strange upregulation of immune checkpoint ligands on monocytes in a simple 2 cell co-culture of monocytes with CD8 T cells?

For different donors, we observed the upregulated expression of immune checkpoint ligands on monocytes when they were cultured with only CD8 T cells (both antigen-specific and non-specific CD8 T...

20 November 2016 2,001 3 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...

19 November 2016 452 4 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.

14 November 2016 1,930 3 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...

13 November 2016 8,784 2 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 3,691 3 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.

08 November 2016 6,565 3 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....

26 October 2016 3,710 1 View