19 Questions 13 Answers 0 Followers
Questions related from Mark O. Pekarsky
Hello, I am wondering if there's a "limit" for subculturing HeLa cells? If yes, what is it? Hom many times they may be subcultured? If there's a limit - WHY is it like this? What's wrong with...
18 August 2016 8,944 3 View
Hello, I am trying to transduce my CD4/CD8 T-cells with lentivector using retronectin from Takara (#T100B) at 50ug/ml, after they are activated by CD3/CD28 for 2 days.The official protocol says...
03 June 2016 6,195 3 View
Hello, I am experiencing problems with expressing GOI2 in GOI1-IRES-GFP:GOI2 cassette in CD4/CD8 cells separated from PBMCs using Miltenyi beads. However expression of the same GOI2 (using the...
27 April 2016 6,793 1 View
Hello dear colleagues, I am experiencing strange problems when performing transDUCTION on my CD4/CD8 cells (separated using Miltenyi beads from total PBMCs from a buffy coat). For now on it is...
19 April 2016 9,476 1 View
Hello everyone, we are trying to transduce T-lymphocytes (TLs) with vector containing IRES, and cannot see any expression using flow cytometry (our target protein is conjugated with GFP). The...
15 March 2016 5,919 3 View
Hello, in my expression cassette I already have 2 proteins that are expressed using IRES. However I'd like to express another protein at the same time (a homing molecule), but, honestly, I do not...
03 February 2016 5,971 3 View
Hello, Could anyone share his experience for how the transduction efficiency of lentivectors may be improved. We have used the spinoculation, protamine sulfate addition. Also just an incubation...
23 July 2015 1,310 6 View
Hi, I received a message from company that if I dissolve 1mg of streptavidin powder (Mw=13300 g/M) in 1ml I will get the "molar concentration" of 19.2 µmol/ml... While if I calculate it myself...
20 July 2015 2,693 2 View
Hi, We would like to inactivate the activity of biotin added to cell culture, but I am wondering what would be the best way to do it... Normally we have a solution of biotin (powder dissolved in...
11 July 2015 6,400 7 View
Hi I am new in the field so I require expert's help :) Briefly the idea of experiment is that the construct (1/2/3) has to be expressed on the surface of the cell, and with the add of component...
25 June 2015 1,768 4 View
Hi all, Sorry I am new to the field, have never performed that assay before. We have T-cells that express a certain receptor on its surface, and we're interested if it can do something to CD19...
19 June 2015 1,437 4 View
Hi, Never thought about it however now I need to ask anyways :) When I use 0.4% Trypan blue for counting of PBMC/lymphocytes (oh btw, does anybody still use Hayem solution today?) I do a 1:1...
13 May 2015 9,500 5 View
Hi, I received a blood bag for PBMC isolation with subsequent T-cells triage required. I received it yesterday evening and put the bag at +4C. I wonder if I can do a ficoll etc NOT the next...
06 May 2015 9,510 8 View
Hello, I am planning to perform a siRNA knockdown using lifetechnologies Lipofectamine RNAiMAX reagent, so taking into account they require 60-80% confluence of cells on the moment of transfection...
02 March 2015 3,541 6 View
Hello, I am trying to do an IP and I am planning to buy some inhibitors (like in the topic, protease/phosphatase/RNAse..). Can you recommend some "good" inhibitors? Thank you!
20 January 2015 1,978 3 View
Hi, I've been using nanodrop for a long time, however I ised it only for RNA/DNA concentration analysis. In fact, there's a "protein" option in nanodrop that I have never used before, though one...
14 January 2015 9,536 19 View
Hello, colleagues. This is my first time I am trying to grow a cell culture from ATCC. So it is Tera-1 (HTB-105) cells and what they recommend on their site is that the medium has to be the McCoy...
18 November 2014 764 3 View
Hello, friends I will need to prepare some total RNA from cells, however I have never did it before. Everyone says that I need to do it in an "RNAse free zone". What that should be in your...
03 November 2014 1,072 3 View
Hi, friends. Would you be so kind to propose the analog of CRL-7800 cell culture (from ATCC)? We badly need these type of cells to have a hiwi expression in it as a positive control (as described...
28 October 2014 6,739 2 View
