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Whole Blood Staining of Human Monocyte Subsets for Flow Cytometry   

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Abstract

This is a general protocol to stain whole human blood for flow analysis with minimal spontaneous activation of monocytes. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.

Keywords: Human, Flow cytometry, Monocytes, Whole blood

Materials and Reagents

  1. Antibodies
    1. Mouse anti-human CD56 FITC (BD Biosciences, catalog number: 340410 )
    2. Mouse anti-human CD2 FITC (BD Biosciences, catalog number: 555326 )
    3. Mouse anti-human CD19 FITC (BD Biosciences, catalog number: 555412 )
    4. Mouse anti-human CD14 PerCP (Life Technologies, Invitrogen™, catalog number: MHCD1431 )
    5. Mouse anti-human CD16 Pac Blue (BD Biosciences, catalog number: 558122 )
    6. Mouse anti-human HLA-DR APC-Cy7 (BioLegend, catalog number: 307617 )

  2. Other materials
    1. Human blood
    2. 10x BD FACS Lysing Solution (BD Biosciences, catalog number: 349202 )
    3. 20% formaldehyde (Tousimis, catalog number: 1008A )
    4. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 20012-027 )

Equipment

  1. Standard Bench-top Centrifuge
  2. 5 ml polypropylene tubes (BD Biosciences, Falcon®, catalog number: 352063 )
  3. BD LSR II flow cytometer
  4. Glass Whole Blood Tube with K3EDTA (BD Vacutainer®, catalog number: 366450 )

Software

  1. FlowJo (Tree Star)

Procedure

  1. Harvest human blood into a 13 ml Glass Whole Blood Tube with K3EDTA and mix the blood by gently inverting the tube several times.
  2. Transfer 100 μl whole blood into 5 ml polypropylene tubes and label the samples accordingly.
  3. Add 5 μl of each antibody into the blood sample and mix by gently tapping the tubes.
  4. Incubate the samples for 15 min in dark at room temperature.
  5. Add 2 ml 1x BD FACS Lysing Solution (10x solution diluted in ddH2O) in each sample.
  6. Vortex sample briefly three times, check for clarity and take to centrifuged within 2-3 min.
  7. Centrifuge 1,200 rpm for 7 min at room temperature.
  8. Remove supernatant and resuspend pellet in 200 μl 2% formaldehyde.
  9. Acquire the samples on BD LSR II flow cytometer.
  10. Analyze data using FlowJo.

Gating strategy:

  1. Gate on monocytes


  2. Gate on HLA-DR+ cells


  3. Exclude CD2+, CD19+, and CD56+ cells


  4. Gate CD16hi, CD14hi, and CD14hiCD16hi monocytes

Acknowledgments

This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research, NY, USA. This work was supported by grants from the NY SLE Foundation (RB), Rheuminations, NIH AI082037 and AR 049938-01, NIH (PO1 AI51392 and the Flow Cytometry and Protein Expression and Tetramer Cores of PO1 AI51392).

References

  1. Liu, Z., Bethunaickan, R., Huang, W., Lodhi, U., Solano, I., Madaio, M. P. and Davidson, A. (2011). Interferon-alpha accelerates murine systemic lupus erythematosus in a T cell-dependent manner. Arthritis Rheum 63(1): 219-229.
  2. Ramanujam, M., Wang, X., Huang, W., Liu, Z., Schiffer, L., Tao, H., Frank, D., Rice, J., Diamond, B., Yu, K. O., Porcelli, S. and Davidson, A. (2006). Similarities and differences between selective and nonselective BAFF blockade in murine SLE. J Clin Invest 116(3): 724-734.
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Liu, Z. (2011). Whole Blood Staining of Human Monocyte Subsets for Flow Cytometry. Bio-protocol Bio101: e69. DOI: 10.21769/BioProtoc.69.
Q&A

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axel broussard
Medical
Human monocyte subsets have got great attention after its initial description in 1989. Its role in health and disease has been described in various publications and still thousands of studies are in the order of their exact determination
10/31/2015 6:01:51 AM Reply
Zheng Liu
The Feinstein Institute for Medical Research

Yes. It has become a very exciting field of research in immunology research.

11/5/2015 11:38:25 AM


hanan fayed
Qena faculty of medicine South Valley University
thanks a lot
I think it should be gating on monocytes instead of lymphocytes
3/4/2015 9:44:59 AM Reply
Zheng Liu
The Feinstein Institute for Medical Research

You are right. Thank you for pointing it out.

11/5/2015 11:37:11 AM


Bio-protocol Editorial Team
bio-protocol.org

Dear Hanan,

Thank you for pointing it out. With Dr. Zheng Liu's permission, "lymphocytes" has been replaced with "monocytes" in the protocol.

11/12/2015 9:44:19 AM


Anna Trier
NIH
Question:

We have been staining monocytes from PBMCs and staining has been working. Recently we switched to whole blood staining for patient samples (so we could take less blood). However, now our classical monocytes (CD14++CD16-) appear to have CD16 expression (compared to the double negative population). Sometimes it is SO bad that there aren't three populations at all just one giant blob that has CD14 expression and one that doesn't (our nonclassical monocytes). Do you have any idea what may cause this? We lyse before we stain...do you think that could be a reason? Also do you know what the double negative population in your CD14 vs CD16 graph could be? It seems like everyone has them regardless of gating method...

Thank you
5/21/2014 12:06:17 PM Reply
Zheng Liu
The Feinstein Institute for Medical Research

Please try to stain before lysis. The double negative cells could be contamination from other population. We try to gate them out using a dump gate containing T, B, NK cell markers, but you always get a few of those cells regardless.

3/9/2015 4:49:54 AM


Chaghaleh M
McGill
My PI asked me to do check the subset of Monocytes after treating with X.
I do not have any experience with Flow Cytometry. I decided to buy Human Peripheral Blood CD14+ Monocytes for example from here:
http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/immune-cells-and-media/human-peripheral-blood-cd14-monocytes.aspx

Then increase them by culturing (I hope they grow like THP-1 cell line), the treat them and then do your protocol.
Is that ok? Do you have any comments?
6/14/2013 8:51:45 AM Reply
Zheng Liu
The Feinstein Institute for Medical Research

Sorry I have no experience in culturing monocytes.

3/9/2015 4:45:40 AM


C Barra
IMIM
Hello have you tried to stain after doing a ficoll?
Do you know if you will find the same 3 populations of monocytes then? Or do you think some marker could be lost? Or any advantage on doing direct blood staining?
Thanks,

Carol

3/28/2013 11:49:23 AM Reply
Zheng Liu
The Feinstein Institute for Medical Research

Yes. You can still find the three populations after ficoll. Whole blood staining has two advantages I can think of.

1. it is easy and saves your time.
2. it doesn't activate monocytes. In my hands, Ficoll activates monocytes which upregulate their surface expression of active form of CD11b and maybe other molecules. So it may not suits certain experiments.

Dr. Z. Liu

3/28/2013 1:45:57 PM


Itshak Golan
Swansea University
Hello,
It is very interesting method; I have two questions;
1. What are the weaknesses of this method? In which conditions it is not good to use it?
2. Do you have experiance with anti-CD44 antibodies?
Thank you in advance for your replay.
Best Regards,
Dr. I. Golan
3/21/2013 11:28:02 AM Reply
Zheng Liu
The Feinstein Institute for Medical Research

I am glad that you thought it was interesting. As to you questions,

1. whole blood staining does not work with all antibodies or cell subsets. For instance, we tried and failed to stain human DC subsets with whole blood staining using a 9 antibody mix. So pilot experiments are needed to determine if your antibodies are compatible with this method.

2. I have no experience with anti-CD44 antibodies.

Hope my answers are helpful.

Regards,

Dr. Z. Liu

3/21/2013 11:41:32 AM


Itshak Golan
Swansea University

Thank you

3/21/2013 3:15:43 PM


This is really helpful to me. I'm wondering if you have experience with CD11b and ly-6c in whole mouse blood. I have noticed that samples from whole mouse blood usually show incomplete lysis of red blood cells, which result in a difficulty in identify monocytes in FSC-SSC picture. Have you tried this? How to solve this problem? Thanks.
1/7/2013 12:00:35 AM Reply
Zheng Liu
The Feinstein Institute for Medical Research

No, I have no experience with CD11b and Ly6C in mouse whole blood. As to RBC lysis, I recommend BD’s PharmLysis which usually results in a pretty complete lysis. But I only used it before staining the cells with antibodies.

1/19/2013 4:53:35 PM


This is excellent. I'm wondering if you have experience with CD86. I've noticed others use CD86 to get all monocytes, then identify the subsets with CD14/16. Have you tried this? Thanks.
6/9/2012 5:03:19 AM Reply
Zheng Liu
The Feinstein Institute for Medical Research

As far as I know, CD86 is an activation marker which is not limited to monocytes. Therefore I personally don’t think you can get all monocytes using CD86.

1/19/2013 4:54:23 PM


can we use cd66 as monocytes surface marker?
4/10/2012 1:34:25 PM Reply
Zheng Liu
The Feinstein Institute for Medical Research

I personally have no experience with CD66. However, CD66 is expressed by many cell types including monocytes, neutrophils, and epithelial cells. So I am not sure how specific it would be as a monocyte marker. In addition, CD14 and CD16 allow us to distinguish the three subsets of monocytes which are phenotypically and functionally different from each other.

I hope this helps. Thank you for your question.

Zheng Liu

4/17/2012 9:55:29 PM


Thanks for the protocol. A found a mistake though. In point 1 of the gating strategy, it should read 'Gate on the monocytes', not lymphocytes.
3/23/2012 12:23:14 AM Reply
Zheng Liu
The Feinstein Institute for Medical Research

Thanks for pointing this out! I have corrected it in the protocol text.

3/23/2012 2:40:56 PM