Presently I am using the following steps:
1. I'm using freshly collected blood in EDTA- vacutainer tube.
2. Then, the blood was dilution at 1:3 ratio (blood: Media) in a cell culture plate at 37oC, 5%CO2 for 2h. Then cells were stimulated with or without LPS (1-10 µg/ml) for 6 to 24 h.
3. Surface staining with CD14, HLA-DR, CD80, and CD86 was performed for flow cytometry.
I am trying to quantify the expression of above mentioned surface markers in monocytes gating. So far I have obtained poor results.
The problem is that I am not getting proper results and non-reproducible results.
Any hints or tips would be well highly appreciated.
Dear Sagar,
Your problem is likely EDTA, try Heparin as the anticoagulant instead, this usually works fine. No need to place the cells in culture media, the blood is a rich source of everything they need. Best regards Christoph
Dear Sagar,
Your problem is likely EDTA, try Heparin as the anticoagulant instead, this usually works fine. No need to place the cells in culture media, the blood is a rich source of everything they need. Best regards Christoph
Agree with Christoph. EDTA removes divalent cations required for inflammatory signalling of LPS to get into cell. Just check various batches of heparin tubes. We found that a lot of heparin tube batches are inflammatory - probably contaminated with pyrogen/endotoxins. Also try to dilute the blood more - I use a 1/10 dilution. I have done serial dilutions of blood and found between 1/5 to 1/10 to work best for LPS activations.
Dear Dr. Edmund Pool,
Thank you for your valuable suggestion.
I will use 1:5 dilution for further experiments.
Dear Sgar: I agree with recomendation to avoid EDTA to work with cells from whole blood,. EDTA IS NOT RECOMENDED FOR CEEL ISOLATION WHEN YOU PLAN TISSUE CULTURE EXPERIMENTS. HEPARIN IS GOOD TO OBTAIN WHOLE BLOOD. Try not use whole blood during stilmulation, instead ISOLATE mononuclear cells using a 1.044 density dradient (Boyum technique), after centrifugation mononuclear cells remain in interface, red blood cells go to bottom, Mononuclear cells are washed twice with your culture media, count cells and add stiumulus as you plan. Good luck.
Dear Dr. Carmona,
Thank you very much for the valuable suggestion.
It has been suggested by different investigators that purification of PBMC from whole blood leads to variations in surface expression of CD14 and CD16 which may lead to incorrect observations involving such purified subsets. Therefore, we adopting whole blood stimulation and analysis of monocyte subtypes in order to infer physiologically meaningful conclusions.
https://www.nature.com/articles/srep13886
The above recommendations to use heparin in the place of EDTA should certainly help.
Unfortunately, there are some downsides to incubating whole blood -- namely, the breakdown products of RBC and their relatively toxicity to cells. One recommendation might be to subject cells to a very brief period of RBC lysis and several rounds of washing before incubation, to remove the majority of the RBCs, debris, and ideally the majority of the platelets.
If you feel you need to use "untouched" whole blood (no gradient, no lysis), I would recommend significantly shortening the incubation times. In the Nature paper you linked, they do not go beyond 6 hours of stimulation. As neutrophils are not removed in whole blood stimulations, and blood-derived neutrophils do not survive well in culture conditions (a half-life of around 6-8 hours), beyond a 6 hour incubation point the large amount of necrotic and apoptotic neutrophils will significantly change both the phenotype and viability of the monocytic cells you would like to examine (based on your panel). LPS stimulation will likely only exacerbate this phenomenon, so perhaps following a time course as layed out in the paper (1, 2, 4, 6 hours may yield more consistent results).
If the end goal is to truly focus in on monocyte responses to LPS stimulation, you might consider a magnetic selection protocol, which usually result in very low background cell activation and will remove the need for density gradient or RBC lysis in most cases.
- You can elect to use positive direct selection, which will likely be the cleanest but will result in the binding of CD14+ site and may necessitate alternative markers: https://www.stemcell.com/easysep-direct-human-monocyte-isolation-kit.html.
- You can also try negative selection, removing all other cell types -- if you are interested in examining "inflammatory" CD16+ monocytes, the best kit is likely: https://www.stemcell.com/products/easysep-human-monocyte-enrichment-kit-without-cd16-depletion.html, which will leave CD16+ expressing cells in the mix.
Best of luck!
Ex Vivo Whole-Blood LPS Stimulation. The concentrations of TNF, IL-6, and IL-1 in blood increase after stimulation with bacterial lipopolysaccharide (LPS).
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