CD14 CD16 staining protocol
General remarks
Fluorochrome-conjugated antibodies used are anti-CD14 (receptor for Lipopolysaccharide = receptor for endotoxin), anti CD16 (low affinity (Type III Fc receptor for IgG) and the respective isotype controls. For CD14 antibodies the LPS receptor blocking type should be employed, since this type of reagent gives the strongest staining. We use My4-FiTC (# 6603511, Coulter). For CD16 many antibodies may work. Some antibodies give weak staining on granulocytes (B73.1). Others show strong staining of granulocytes (DJ130c, NKP15). Depending on the conditions either type of antibody may lead to interference with the CD14+ CD16+ monocyte staining. We use Leu11c-Phycoerythrin (B73.1 # 347617 Becton Dickinson). Isotype controls should be obtained from the same source in order to assure a comparable method of immunoglobulin purification and of fluorochrome conjugation for control and the specific reagent. Isotype controls are used at the same final immunglobulin concentration as the specific antibody. All reagents are titrated in serial twofold dilutions in order to determine a proper dilution in the plateau of the titration curve.
Staining of cells
Staining can be done either on isolated peripheral blood mononuclear cells (PBMC) or on whole blood. For both approaches the following combinations are set up:
anti CD14 FITC - anti CD16 Phycoerythrinanti CD14 FITC - isotype Phycoerythrin isotype FITC
anti CD16 Phycoerythrin
For whole blood staining EDTA-anticoagulated blood is stored on ice. It is carefully mixed and an aliquot is used for determination of the leukocyte count. 100 µl aliquots are then given into three 5 ml round bottom tubes (for example Falcon # 2008). To deplete the plasma 2 ml of phosphate buffer saline without Ca ++ and Mg ++ (PBS) is added to each tube, the tubes are spun for 5 min. at 400 g and supernatant is carefully removed. To the pellet antibodies are added, samples are carefully mixed and are put on ice for 20 min with light protection. Samples are then lysed and fixed. We use the Coulter Q prep reagents together with the Q prep workstation. After this process is complete 1 ml of water is added and this is followed by 2 ml of PBS. Samples are mixed and spun for 5 min at 400 g. The supernatant is removed and cells are resuspended with 0,5 ml PBS to be analysed within 2 hs.
For staining of peripheral blood mononuclear cells (PBMC) 10 6 cells each are given into Eppendorf tubes these are spun for 3 min at 400g, the supernatant is carefully removed and replaced by 50 µl of appropriately diluted antibody. Samples are mixed and put on ice for 20 min. Samples are washed twice with 1,0 ml of PBS, the tubes are spun for 5 min at 400 g and the cells are then resuspended in 0,2 ml PBS to be analyzed within 2 hs.
Data acquisition
FACS analysis begins with collecting 10 000 events with the gates set around all leukocytes in the forward angle light scatter (FALS) - 90° light scatter (90° LS) histogram. This histogram is later used to determine the proportion of monocyte gate cells among all leukocytes in order to calculate the absolute numbers of monocyte subpopulations.
Next a gate is set around monocytes which may look like this:
monogate for PBMC
monogate for whole blood
The monogate should include some of the lymphocytes and in whole blood staining it should avoid the granulocytes. Please note that frequently additional events are seen to the right of the monocytes. These are monocyte aggregates which can be dispersed by passing the cell suspension through a 0,4 mm needle using a 1 ml disposable syringe.
With gates on monocytes the PMTs and the compensations are set such that the following 2 color histograms are obtained.
Fig 3 ( Donor HWLZH, 2.5.96)
With this FACS set-up 10 000 events (or more) are collected per sample.
Analysis of collected data
Aided by the two control stainings # 2 and # 3 the gates are set around around all monocytes like this:
Fig 4
These are set as 100%. The percentage of CD14 + CD16 + and of CD14 ++ cells is determined with the following gates :
Fig 5
and the percentages among all monocytes are calculated.
In the example given there are a few cells that locate between the 2 gates in Fig 5. They have been included in the determination of all monocytes (Fig 4) but they were added neither to the CD14 + CD16 + nor to the CD14 ++ . These cells are CD14 ++ and do express some CD16 (CD14 ++ CD16 + cells). They account for about 1,5 % of all monocytes in this example and likely represent a transitional form. These cells may also be enumerated separately.
Calculation for PBMC
In order to calculate the absolute counts of monocyte subpopulations among PBMC the percentage of cells in the monocyte gate among all PBMC is determined by projection of the gate into the light scatter histogram for all PBMC. This value is then used for the following calculation:
Percentage of population x Percentage of monocyte in monocyte gate gate cells among all PBMC -------------------------------------------------------------- 100 = Percentage of monocyte population among all PBMC
Example:
Calculation for whole blood
In order to calculate the absolute count of monocyte subpopulations among all blood leukocytes in whole blood analysis the percentage of monocyte gate among all leukocytes is determined by projection of the monocyte gate into light scatter histogram of all leukocytes. This percentage is then used for the following calculation:
Percentage of population x Percentage of monocyte in monocyte gate gate cells among all leukocytes -----------------------------------------------------------------------= 100 = Percentage of population among all leukocytes
Example:
Using the leukocyte count that was initially determined for whole blood the absolute count of monocyte subpopulations can be calculated. In the example with 0,56% CD14 + CD16 + monocytes among all leukocytes and a leukocyte count of 9000 / µl the CD14 + CD16 + cells are calculated to be:
Comment: In average CD14 + CD16 + monocytes are about 50 cells per µl in healthy donors but data on large numbers of donors and on the influence of age and gender are lacking. CD14 + CD16 + monocytes have been found increased in a variety of inflammatory and infectious diseases including sepsis (sometimes > 500 cells/µl), HIV infection and tuberculosis. Also these cells increase with injection of lipopolysaccharide and after M-CSF therapy.