- Markus Schäfer added an answer:5Does anyone have experience with (Amaxa Nucleofector - based) transfection of murine hematopoietic progenitor cells?
I want to transfect murine hematopoietic progenitors with a Cas9-encoding plasmid (so the thing is quite large).
I want to use Pax5- or Ebf1-deficient cells, either freshly isolated and in vitro cultured for 5 days or some cryopreserved cells. The cells are somewhat like CLP (ALP/BLP) - like in a way that they have multi-lineage (myeloid, T-, B-cell,...) potential.
We have a Amaxa nucleofector (Lonza) in the lab and it seems that this is the best way to get DNA in those cells. However, the culture looks like a graveyard 24h post transfection.
Therefore, I was wondering if you have any suggestions regarding nucleofector protocolls/kits for such cells.
I used Bcl2 transgenic cells progenitors and nucleofected them in buffer V or L according to the manual with the programs X-01 or X-05. The plasmid that I am transfecting is rather large (pSpCas9(BB)-2A-GFP (PX458) from Dr. Feng Zhang's lab ~kb). In non-Bcl2 transgenic cells I have hardly any GFP+ cells. About 10 - 25 % of the Bcl2+ cells are in the life gate 24 h post nucleofection. Of those 10 - 40 % are GFP+.
I would test these conditions (Buffer V/L & programs X-01/5 individually). For me L and X-01 worked best. I also tested the human CD34+ kit with the program U-08 from Lonza, but it was not as efficient as the other buffers).Following
- Cédric Sapet added an answer:3Can anyone help me with the spheroid formation assay for mouse endothelial cells?
I wanted to perform a spheroid angiogenesis assay for my endothelial progenitor cells. I have been told to seed 500-1000 cells in serum free medium in non-adherent 96 well plates. After incubation for approximately 24 hours the cells will adopt a spheroid configuration and then I should just overlay 100 µl matrigel over the spheroid and incubate for further time till sprouting occurs. I just want to know if this will work or can anyone provide any protocols which do not involve the use of methocel.
please forgive for not providing a protocol for your specific application and to answer in a more "commercial" way but I would like to propose you some transfection reagents you may be interested in. Actually, we have developped transfection reagents specifically for 3D cultures for both DNA and siRNA.
3D-FectIN (for gels and hydrogels) works very well with matrigel and endothelial cells (cells get transfected and keep their ability to form microtubes) and maybe it could be a valuable tool for your research. Briefly, complexes of nucleic acids and 3DFectIN are formed and mixed with Matrigel. Cells are then added and become transfected while invading the gel. You can find more information in the link below.
If you want to try this reagent please feel free to contact me directly at firstname.lastname@example.org or via Researchgate.
Good luck for your experiment,
- Nicol Poncina added an answer:11What type of cells are these? I attached a photo and wonder are they endothelial progenitor cells?
what do you think about these cells? i'm attending to isolate epc..
What was the medium you cultured them in? EGM-2 .
does it have the essential cocktail of growth factors? yes
how do you isolate them? do you use a fibronectin coating for the plate?
- Meijie Qu asked a question:OpenHow do I determine the oxygen glucose deprivation time of endothelial progenitor cells?
Hello, I want to ask you a question how to determine the OGD time of EPC. I have read some articles about this. But I found 16 hours and 48hours were used. （Please forgive me that I did not find more article about EPCs OGD in pubmed) Another article named Adaptation to oxygen deprivation in cultures of human pluripotent stem cells, endothelial progenitor cells, and umbilical vein endothelial cells demonstrated the dissolved oxygen levels of both EPCs and HUVECs equilibrated with the exposure concentration after 12h ischemia. Does it mean that we need progress OGD at least 12h.
Can you kindly tell me the hours or recommend articles to me? Thank you for your kind consideration.Following
- Kondababu Kurakula added an answer:9What is a suitable protocol to isolate rat lung (microvascular) endothelial cells?
We are interested in isolating and culturing rat lung microvascular endothelial cells. Any help is highly appreciated.
Thank you Abha Sahni for your kind reply.Following
- Yvan Devaux added an answer:1Does DMSO have an influence on tube formation assay (with EPC)?
Does DMSO have an influence on tube formation assay (with EPC) ?
DMSO can affect any cell function, depending on its concentration. The trick is to have the proper control.Following
- Ebba Brakenhielm added an answer:6Which is the best protocol to isolate mouse endothelial cells (prefably from the heart or aorta)?
Hi all, I would like to use some mouse endothelial cells (ECs) for my experiments, but so far have failed to get any of those cells growing, even from those commercial one bought from Cell Biologics (the only company that seems to be selling mouse ECs).
Alas, I would like to find a way to grow them in-house and would like to seek your help on any good protocols on isolating mouse ECs (prefably ECs from aorta and heart), and what is the success rate of such protocol?
Thanks in advance!
Characterization of microvascular endothelial cells isolated from the dermis of adult
mouse tails Microvascular Research 82 (2011) 97–104
Nicely describes magnetic activated cell sorting (MACS) and further culture applicable to ECs of any origin.Following
- Carlos Bueno-Betí added an answer:2Are endothelial progenitor cells (EPCs), immunoprivileged?
I want to run some experiments with Human EPC in Rat bone defect. I prefer to use Normal Rat rather than Athymic Rat. However, I am not sure about EPC's immunoprivilege character.
I would recommend the use of nude rats. If have done matrigel plug assay on nude mice with good results.Following
- Einari Aavik added an answer:3Can anyone help with a list of Endothelial specific genes for microarrays?I want to compare primary HUVECs with my immortalized HUVECs. I have done microarrays and was wondering if I can somewhere (some database etc) find a list of cell type specific genes (which, in my case, would be endothelial cell specific genes). Any kind of help would be appreciated.Hi Muhammad!
There is an article from Patrick Brown's lab from 2003:
Chi JT1, Chang HY, Haraldsen G, Jahnsen FL, Troyanskaya OG, Chang DS, Wang Z, Rockson SG, van de Rijn M, Botstein D, Brown PO.
Endothelial cell diversity revealed by global expression profiling.
Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10623-8.
Then you may take a closer look at Gene Omnibus microarray dataset GSE3239. There they have identified genes more abundant in endothelial cells (EC), but low in smooth muscle cells (SMC), fibroblasts and epithelial cells. The list below represents fold differences between mRNA expression levels in EC and SMC:
Gene ArterialEC VenousEC
SOX17 1,21 16,87
MYRIP 1,63 10,54
SRGN 1,72 1,81
LIPG 1,95 9,65
ALDH1A1 1,97 19,29
IGF2BP3 2,29 5,76
SEC14L1 2,57 4,49
TEK 2,83 8,61
PROCR 2,92 1,96
ITM2A 2,95 23,07
ESM1 3,43 2,11
ARHGDIB 3,77 5,00
HOXB7 4,71 3,73
PTPRB 5,36 9,58
THBS1 5,48 5,47
MYCT1 5,87 9,37
HECW2 6,44 9,76
RASIP1 7,67 9,72
CALCRL 8,31 35,38
PGF 8,46 4,14
PCDH1 8,50 19,51
BST2 8,81 0,81
TINAGL1 9,26 6,39
NRN1 10,02 14,73
CGNL1 10,24 17,49
IFI27 10,44 3,30
HCLS1 11,14 10,54
ANGPT2 12,14 10,91
EDN1 12,15 19,46
GIMAP7 12,17 23,84
IL1RL1 12,61 18,68
MMRN1 13,13 152,78
C17orf72 13,31 7,66
IFI27 13,93 3,57
MLZE 14,37 20,91
RAPGEF5 14,40 31,24
AL576606 14,50 1,31
EMCN 14,88 18,24
FRMD4B 15,06 18,14
MMRN1 15,38 150,30
GATA3 15,66 2,35
ABI3 16,02 19,85
PTPRE 16,35 33,37
LYL1 16,82 29,30
FGD5 16,97 34,99
EGFL7 18,16 67,55
ESAM 18,65 20,07
ASRGL1 19,40 38,06
SHE 19,44 35,15
RNASE1 19,53 94,28
CLDN5 20,28 48,54
BI823044 20,45 152,97
CDH5 20,84 183,71
CLEC1A 21,14 60,02
GIMAP6 22,08 119,60
MPZL2 22,84 219,25
PRKAR2B 23,02 42,90
TIE1 23,26 77,59
ERG 23,90 61,29
LAPTM5 24,13 58,32
FBP1 24,32 28,03
TM4SF18 25,84 53,04
BQ717190 26,35 37,22
MGAT4A 28,26 143,79
GIMAP4 28,97 225,52
AA480009 29,10 61,80
ICAM2 29,60 61,18
FAM124B 30,65 27,04
NMNAT1 31,47 50,28
CYTL1 31,51 175,05
BMX 32,43 229,34
C10orf58 44,67 80,90
FAM107A 49,62 225,09
CXorf36 67,49 85,36Following
- Dirk Henrich added an answer:2Are these cells EPCs or not?Attached is the picture of putative EPCs growing on fibronectin coated plates for 13 days. Do the cells look like EPCs? I am asking this because I am having trouble in staining them with CD31. Is staining EPCs different from staining endothelial cells?Dear Maulasri,
my answer comes maybe too late. Those cells look like the so called early EPC which develope from monocyte precursors. Those cells are not stem cells but they express some endothelial markers such as CD31, they should incorporate Dil-ac-LDL and express vWF. Although no stem cells, those cells work pretty fine in vivo. We observed a much accelerated wound healing and a significant improved bone healing in our animal models.
To identify those cells you have to combine several markers. They should express all markers mentioned above simultanously. This can be proved easily by flow cytometry.
- Santiago Roura added an answer:22Unable to passage Endothelial progenitor cells.I have isolated Endothelial progenitor cells from mice bone marrow and grown them for 15-20 days on fibronectin. After 16 days, I need to passage them as to use the cells for FACS or re-seed them. But when I try to passage them with 0.05% trypsin, they become round but don't detach from the surface and I end up getting dead cells, debris and differentiated cells. Please suggest a convenient method to split them so that I can use them for FACS and re-seeding without losing their stem cell properties?Dear colleagues,
Be aware that the identification of ture vascular precursors has been a matter of debate for the past 15 years since the publication of the study Asahara (Science 1997) who described putative endothelial progenitors. Subsequently, diverse cell subpopulations have been studied as colony forming units-Hill (Hill NEJM 2003), circulating angiogenic cells or CAC (e.g. described by Dimmeler group, Vasa Circulation 2001) and endothelial colony-forming cells as those studied by Yoder (Yoder Blood 2007) that varied in terms of phenotype, contribution to vascular homeostasis and purity. In addition, cell isolation and culture techniques are different for each one subpopulation.
If it helps, we have recently published an extensive revision regarding this strong controversy (Roura J Vas Res 2013).Following
- Rajesh Mohandas added an answer:3Does anybody have any experience with detection and quantitation of circulating EPCs in mice?I want to detect EPCs in peripheral blood of mice treated with an inhibitor of CXCL12You can enumerate circulating EPC by flow. The exact markers which define epc are controversial. I would use something which has been published for the type of work you are doing.http://www.jci.org/articles/view/33125 I think this is very similar to what you are looking for. You can also enumerate colonies from peripheral blood. Again there are early and late EPCs. Late EPCs are thought to be more true EPCs. However there is some controversy on that as well.Following
- Dewi Sukmawati added an answer:10EPC cells could not be trypsinizedI isolated endothelial progenitor cells (EPCs) from mice bone marrow and cultured them in a human fibronectin coated plate for 4 weeks. The cells grew well in the plate albeit there were some other cells except EPCs. But the cells didn't respond to the trypsin (0.25%) when I tried to typsinize them. Some of the cells could be detached while most of the cells were still on the plate although their morphology became round. Is there any method other than trypsinization I could use to passage my cells, such as degrading the fibronectin?Following
- Sylvain Fraineau asked a question:OpenECFCs (Endothelial Colony Forming Cells) or EOC (Endothelial Outgrowth Cells) = HUVECs?I've just read a good article from Tura O published in Stem Cells.
It states that EOC (quite the same as ECFCs) do not differ from mature endothelial cells and may be derived from a vascular source outside the bone marrow.
Has anyone found the same results?Following
- Badri L Aekbote added an answer:16Coating the surface of endothelial cells with fluorescent dye?I would like to coat the surface of endothelial cells (fixed on glass slide) with streptavidin cojugated dye specifically or non specifically?@George
thanks for your reply and suggestion, actually my aim is to have a sheet of endothelial cells on glass slide whose cell surface are stained with fluorescent dye, preferably above 550nm, i tried surface functionalization of these cell using these Biotin-bsa and NHS biotin, i want to use these slides eventually for Metal enhanced fluorescence , i know the method iam using is quiet unspecific and i found this method is killing the cells at the end ( i use pbs buffer for functionalization plus incubation at 4C)
to you have any suggestion how can i improve with this protocol of using either bBSA or NHS biotin? or best is that i should use the antibodies as u mentioned
i hope i could make my point
About Endothelial Progenitor Cells
For all scientists involved in endothelial progenitor cell research.