One step phagocytosis-cell activation-cell death assay

Abstract

The invention relates to methods to evaluate in one single assay the biocompatibility of materials based on the simultaneous determination of the phagocytosis, cell activation and cell death produced by said materials, preferably, in peripheral blood or other human cells and proximal fluids. The invention also relates to a kit to perform the method of the invention.

Claims

1. A method for simultaneous evaluation of cytotoxicity, immune modulation and phagocytosis induction of a fluorescently-labeled material, said material being a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where one or more of its external dimensions comprises a size ranging from 0.001 m to 1000 m, comprising the following steps: a) incubating a biological sample with the fluorescently-labeled material, giving rise to stimulated cell populations; b) incubating the stimulated cell populations from the biological sample of step a) with the following molecules fluorochrome-labeled antibodies specific for monocytic markers, T-CD4.sup.+ lymphoid markers, T-CD4.sup. lymphocyte-associated markers and markers of molecules involved in immune modulation, an inhibitor of protein secretion, and viability cell markers, wherein each marker and the fluorescently-labeled material is detected with a different fluorochrome, c) passing the cell populations resulting from carrying out step b) through a flow cytometer and measuring the different fluorescent emissions in a single measurement; and d) counting the number of viable cells, activated cells and cells that have phagocytosed the fluorescently-labeled material within each cell population by measuring the amount of fluorescent signal associated with every individual cell and to every individual immune modulation involved molecule marker, viability marker and phagocytosed particle marker used.

2. The method according to claim 1, wherein the fluorescently-labeled material is a nanomaterial or a micromaterial.

3. The method according to claim 1, wherein the biological sample is selected from the group consisting of peripheral blood, ascitic fluid, pleural effusion, cerebrospinal fluid, bone marrow, lymph node, lymph fluid, synovial fluid, a single cell suspension prepared from a solid tissue and cell lines.

4. The method according to claim 1, wherein the monocytes, T-CD4.sup.+ lymphocytes and T-CD4.sup. lymphocytes markers are selected from the group consisting of CD14, CD4, CD3, CD45 and any combination thereof.

5. The method according to claim 1, wherein the viability cell markers are selected from the group consisting of Annexin V, Propidium Iodide (PI); 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI); 7-amino-actinomycin D (7-AAD); TO-PRO-3 and DNA-binding dyes.

6. The method according to claim 1, wherein the markers of molecules involved in immune modulation are selected from the group consisting of Tumor Necrosis Factor alpha (TNFalpha), Interferon gamma (IFN), Interleukin 6 (IL6), Interleukin 12 (IL12), Tumor Necrosis Factor beta (TNF), Interleukin 2 (IL2), Interleukin 8 (IL8), Interleukin 10 (IL10), Interleukin 13 (IL13), Interleukin 16 (IL16) and any combination thereof.

7. The method according to claim 1, wherein the fluorochromes are selected from the group consisting of fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), allophycocyanin (APC), Alexa fluor 488, Alexa 647, Alexa 710, Alexa fluor 405, cyanin 5 (Cy5), Cyanin 5.5 (Cy5.5), pacific blue (PacB), horizon V450 (HV450), pacific orange (PacO), brilliant violet (BV), Horizon V500, 00515, Krome Orange, CF Blue, quantum dots or conjugates thereof coupled to PE, APC or PerCP, and any combination thereof.

8. The method according to claim 1, wherein the inhibitor of protein secretion is a metalloprotease inhibitor.

9. The method for analyzing the biocompatibility of a fluorescently-labeled material, said material being a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where one or more of its external dimensions comprises a size ranging from 0.001 m to 1,000 m, comprising: measuring the cytotoxicity, immune modulation and phagocytosis inducing properties of the fluorescently-labeled material by a method according to claim 1, wherein a lower degree of cytotoxicity, immune activity, and/or phagocytosis function than a control profile obtained by the same method but without the presence of the fluorescently-labeled material, is indicative that the fluorescently-labeled material is biocompatible.

10. The Method according to claim 9, wherein the fluorescently-labeled material is a nanomaterial or a micromaterial.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1Precise discrimination of activated cells (showing higher levels of expression of TNFalpha), upon stimulation with LPS plus IFNgamma, can be performed comparing to an unstimulated control used as negative control (Panel A) to set the threshold above which TNFalpha positive cells can be identified. The gating for TNFalpha positive CD4.sup.+ and CD4.sup. T lymphocytes (CD4.sup.+ T Ly and CD4.sup. T Ly, respectively) was performed in a TNFalpha-CD4 dot plot (Panel B). In the case of monocytes, a TNFalpha-CD14 dot plot was employed (Panel C).

(2) FIG. 2Precise discrimination of activated cells (showing higher levels of expression of TNFalpha), upon stimulation with PMA plus ionomycin, can be performed comparing to an unstimulated control used as negative control (Panel A) to set the threshold above which TNFalpha positive cells can be identified. The gating for TNFalpha positive CD4.sup.+ and CD4.sup. T lymphocytes (CD4.sup.+ T Ly and CD4.sup. T Ly, respectively) was performed in a TNFalpha-CD4 dot plot (Panel B). In the case of monocytes, a TNFalpha-CD14 dot plot was employed (Panel C).

(3) FIG. 3Populations of interest (monocytes and lymphocytes T) can be detected by accurate flow cytometry gating strategies after staining for expression of CD3, CD4, CD14, and CD45. In the presented staining, first gating on the diagonal of FSC-H and FSC-A is performed (Panel A) to eliminate the cell debris and the cell aggregates followed by gating based on positivity for CD45 (Panel B). Monocytes and T lymphocytes can be accurately identified gating on CD14 expressing cells vs. CD3 expressing cells, respectively (Panel C). The T lymphocytes selected can be further evaluated with the CD4 marker, resulting in two subsets: CD4.sup.+ T lymphocytes and CD4.sup. T lymphocytes (Panel D).

(4) FIG. 4Results of viability analysis after incubation of PBMN cells with carboxylate-modified polystyrene fluorescent yellow-green latex beads for 4 hours. Staining for annexin V allowed the distinction between apoptotic cells (positivity for annexin) and viable cells (negativity for annexin). The gating for annexin positive CD4.sup.+ and CD4.sup. T lymphocytes (CD4.sup.+ T Ly and CD4.sup. T Ly, respectively) was performed in an annexin-CD4 dot plot (Panel A). In the case of monocytes, an annexin-CD14 dot plot was employed (Panel B).

(5) FIG. 5Results of immune system stimulation analysis after incubation of PBMN cells with carboxylate-modified polystyrene fluorescent yellow-green latex beads for 4 h at a ratio of 10 beads per cell. Staining for TNFalpha allowed the distinction between activated cells (positivity for TNFalpha) and non-activated cells (negativity for TNFalpha). An unstimulated control without beads can be used as a negative control (Panel A) to set the threshold above which TNFalpha positive cells can be identified. The gating for TNFalpha positive CD4.sup.+ and CD4.sup. T lymphocytes (CD4.sup.+ T Ly and CD4.sup. T Ly, respectively) was performed in a TNFalpha-CD4 dot plot (Panel B). In the case of monocytes, a TNFalpha-CD14 dot plot was employed (Panel C).

(6) FIG. 6Results of phagocytosis analysis after incubation of PBMN cells with carboxylate-modified polystyrene fluorescent yellow-green latex beads for 4 h at a ratio of 5 beads per cell. The fluorescence detected in the FITC channel (corresponding to the fluorescent yellow-green marker of the beads) allows the precise discrimination between phagocytic and non-phagocytic cells. In an FITC-CD14 dot plot is possible to recognize with high accuracy those phagocytic monocytes (CD14.sup.+/FITC.sup.+) from those non-phagocytic ones (CD14.sup.+/FITC.sup.). The same strategy is performed for phagocytic and non-phagocytic T lymphocytes (CD14.sup./FITC.sup.+ and CD14.sup./FITC.sup., respectively).

(7) FIG. 7Strategy of analysis of phagocytosis for non-fluorescent microparticles. In the left, two dot plots showing the gating of phagocytic cells by two different but comparable strategies. Detection of positive cells in the FITC channel (Panel A) corresponding to cells phagocyting fluorescent yellow-green beads. In Panel B, gating of cells with high SSC corresponding to the same cells gated in Panel A. Therefore, for non-fluorescent beads, the FSC-SSC dot plot (Panel C) can be used for identifying the phagocytic cells by using the threshold set for fluorescent beads (Panel B).

EXAMPLES

Example 1Simultaneous Multiparametric Flow Cytometry Analysis of Viability and Immune Response to Different Stimuli

(8) 1.1 Material and Methods

(9) Heparin anti-coagulated peripheral blood (PB) samples were obtained from 2 healthy individuals and processed within the first 2 hours after collection for simultaneous analysis of viability and immune response to distinct stimuli (i.e. activation of T lymphocytes and monocytes, measured by the expression of TNFalpha).

(10) 1. 2. Sample Preparation

(11) Peripheral blood mononuclear cells (PBMN cells) were purified by density gradient centrifugation using Biocoll Separating Solution (Biochrom, Germany), according to the manufacturer's protocol. After centrifugation, the PBMN cells interphase was collected and quantified using a Neubauer chamber. The PBMN cell culture was carried out in RPMI-1640 media supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin and 40 M TACE inhibitorTAPI-2(Sigma-Aldrich, St. Louis/MO, USA) in a 24-well plate (2 cm.sup.2 diameter). TAPI-2 was used as secretion blocking agent for detection of membrane-bound TNFalpha. Alternatively, 10 g/mL of Brefeldin A (Sigma-Aldrich) was used to inhibit cytokine transport from the endoplasmic reticulum to the Golgi apparatus, instead of the TACE inhibitor, as a control condition for TAPI-2. In these control conditions, TNFalpha expression was assessed intracellularly. For stimulation purposes 100 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich) plus 10 ng/mL IFNgamma (Promega, Madison/WI, USA) (FIG. 1) or 10 ng/mL of phorbol 12-myristate 13 acetate (PMA) (Sigma-Aldrich) plus 0.75 g/mL of ionomycin (Sigma-Aldrich) (FIG. 2) were added, to stimulate (i.e. induce cytokine secretion) monocytes and T lymphocytes. As a basal activation control, cells were incubated without the different stimuli. The PBMN cells were incubated in darkness at 37 C. in 5% CO.sub.2 atmosphere for 4 hours.

(12) After this incubation period, cells from each well were collected in a tube using phosphate buffer saline solution (pH 7.4) (PBS). Adherent cells were detached by gentle scrapping. Then, cells were centrifuged at 400 g for 10 minutes. The supernatant was discarded and the cell pellet was resuspended in 2 mL of PBS. Another centrifugation (400 g, 10 minutes) was performed and cells were finally resuspended in 0.1 mL of PBS solution.

(13) For those conditions incubated with TAPI-2, surface membrane staining was performed to identify the distinct populations of interest and assess the expression of TNFalpha. Therefore, each sample was stained with the following combination of monoclonal antibodies. The specificity of the monoclonal antibodies used, their origins and their fluorochromes were as follows: CD3 APC-H7 (Clone SK7); T-lymphocyte marker (BD, San Jose/Calif., USA) CD4 PE (Clone HP2/6); cytotoxic T-lymphocyte marker (ImmunoStep, Salamanca) CD14 PE-Cy7 (Clone RM052); monocyte marker (BD, San Jose/Calif., USA) CD45 PerCPCy5.5 (Clones 2D1); pan-leucocyte marker (BD, San Jose/Calif., USA) TNFalpha APC (Clone MAb11); immune response marker (BD, San Jose/Calif., USA)

(14) The tubes were gently vortexed and incubated in the dark for 15 minutes at room temperature. Immediately after this incubation period, 2 mL of PBS was added to each tube followed by gentle vortex and a centrifugation at 400 g for 5 minutes. The supernatant was discarded and a new washing step with 2 mL of PBS was performed. Afterwards, 0.2 mL of 1 annexin V binding buffer was added followed by the annexin V-CFBlue apoptosis marker (ImmunoStep, Salamanca/Spain). Cells were incubated for 15 minutes at room temperature and were stored at 4 C. in the darkness until analyzed in the flow cytometer.

(15) For those activation control conditions incubated with Brefeldin A, surface membrane staining was performed with monoclonal antibodies aimed at the identification of populations of interest and an intracellular staining was made for the assessment of TNFalpha production.

(16) The specificity of the monoclonal antibodies used, their origins and their fluorochromes were as follows: CD3 APC-H7 (Clone SK7); T-lymphocyte marker (BD, San Jose/Calif., USA) CD4 PE (Clone HP2/6); cytotoxic T-lymphocyte marker (ImmunoStep, Salamanca) CD14 PE-Cy7 (Clone RM052); monocyte marker (BD, San Jose/Calif., USA) CD45 PerCPCy5.5 (Clones 2D1); pan-leucocyte marker (Becton Dickinson Biosciences, BD, San Jose/Calif., USA) TNFalpha APC (Clone MAb11); immune response marker (BD, San Jose/Calif., USA)

(17) For staining procedure, CD3 APC-H7, CD4-PE, CD14 PE Cy7 and CD45 PerCP Cy5.5 monoclonal antibodies were added and the cells were incubated for 15 minutes at room temperature in the darkness. Cells were washed with 2 mL of PBS and centrifuged for 5 minutes at 400 g and the supernatant was discarded. Cells were then fixed with 100 L of reagent A (fixation solution; Fix&Perm, An der Grub, Vienna, Austria) and incubated for 15 minutes in the dark at room temperature. After this period, 2 mL of PBS was added and the suspension was centrifuged for 5 minutes at 400 g, and the supernatant was discarded. Cell pellet was resuspended by mixing gently and 100 L of Reagent B (permeabilizing solution; Fix&Perm) was added simultaneously with the antibody against the intracellular protein (TNFalpha). The tubes were gently vortexed and incubated in the dark for 15 minutes at room temperature. After this period, cells were washed twice with 2 mL of PBS (5 minutes at 400 g) and cells resuspended in 0.3 mL of PBS for acquisition.

(18) 1.3. Data Acquisition and Analysis

(19) Data acquisition was performed on a FACSCanto II flow cytometer (Becton Dickinson Biosciences, BD, San Jose/Calif., USA) using the FACSDiva software (v6.1; BD). For data analysis, the Infinicyt software (Cytognos SL, Salamanca, Spain) was used.

(20) Data analysis was performed in four steps. First, we selected the cells of interest (white blood cells) and excluded cell debris and cell aggregates, based on the expression of CD45 and light dispersion characteristics (forwards side scatterFSCand sideward side scatterSSC). Secondly, we identified and selected the monocytes (CD14.sup.++/CD4.sup.+/CD3.sup./CD45.sup.+), the T-CD4.sup.+ lymphocytes (CD45.sup.++/CD3.sup.+/CD4.sup.+/CD14.sup.), and the T-CD4.sup. lymphocytes (CD45.sup.++/CD3.sup.+/CD4.sup./CD14.sup.) (FIG. 3). Thirdly, for those tubes labelled with Annexin V, apoptotic cells were identified within each population as those cells in which Annexin V-CFBlue signal was detected (FIG. 4). Afterwards, non-annexin positive cells (corresponding to living cells) were used for determining TNFalpha-APC positive cells (for measuring the immune response). Both in the control conditions incubated with TAPI-2 (for plasma membrane detection of TNFalpha) and those conditions incubated with Brefeldin A (for intracellular detection of TNFalpha), the effect of the stimulation in each cell population was assessed on unstimulated samples as a control and TNFalpha production was based on percentage of positive cells, after subtracting the percentage of cells staining above the threshold for positivity in the negative control.

(21) 1.4. Results

(22) The usage of PMA plus ionomycin activation stimuli during 4 hours, in presence of TACE-inhibitor, lead to significant apoptosis induction in monocytes, CD4+, and CD4.sup. T cells (89.010.9%, 35.42.8% and 51.71.7% Annexin V positive cells, respectively), compared to the unstimulated controls (13.13.7%, 14.92.4% and 24.16.3%, respectively). This was also observed, in less extent, also for LPS plus IFNgamma stimuli (80.414.9%, 15.92.0% and 24.61.5% Annexin V positive cells, respectively).

(23) Activation of monocytes, CD4.sup.+ and CD4.sup. T cells was observed as a result of incubation of PBMN cells with PMA plus ionomycin in presence of TACE inhibitor (62.50.5%, 30.119.7% and 7.61.7% TNFalpha positive cells, respectively), compared to the unstimulated controls (2.52.2%, 1.51.4%, and 1.91.8%, respectively). Conversely, stimulation with LPS and IFNgamma only induced significant activation on monocytes vs. CD4.sup.+ and CD4.sup. T cells (81.68.7% vs. 1.60.8% and 2.21.7% TNFalpha positive cells, respectively). A similar pattern was observed for those cases in which inhibition of TNFalpha release was performed using Brefeldin A, with 55.011.8%, 66.79.5% and 35.01.5% TNFalpha positive cells, observed for monocytes, CD4.sup.+ and CD4.sup. T cells stimulated with PMA plus ionomycin; 97.92.0%, 13.412.3% and 14.314.7% TNFalpha positive cells, respectively when LPS and IFNgamma were used as stimuli, all compared to the unstimulated control (32.722.6%, 13.812.8% and 15.715.3% TNFalpha positive cells, respectively).

(24) 1.5. Conclusion

(25) By one single assay, it is possible the simultaneous characterization of activated lymphocytes and/or monocytes cells against an antigen stimulus; in addition, this assay allows the quantitative discrimination between activated vs non-activated cells; among others cells presented in a complex biological sample (i.e. peripheral blood) by immunophenotypic analysis.

Example 2Multiparametric Analysis of Phagocytosis, Viability, and Immune Response after Incubation with Fluorescent Beads

(26) 2.1. Material and Methods

(27) Peripheral blood (PB) was obtained by venous puncture from 2 healthy individuals, placed in lithium heparin tube and processed within the first 2 hours after collection for simultaneous analysis of viability, phagocytosis and immune response to incubation with fluorescent beads.

(28) 2.2. Sample Preparation

(29) Peripheral blood mononuclear cells (PBMN cells) were purified by density gradient centrifugation using Biocoll Separating Solution (Biochrom, Germany), according to the manufacturer's protocol. After centrifugation, the PBMC interphase was collected and quantified using a Neubauer chamber.

(30) A total of 0.210.sup.6 PBMN cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin and 40 M TACE inhibitorTAPI-2(Sigma-Aldrich, St. Louis/MO, USA) in a 24-well plate (2 cm.sup.2 diameter). TAPI-2 was used as secretion blocking agent to reveal the levels of TNFalpha. The sample was incubated with 110.sup.6 and 210.sup.6 latex beads (carboxylate-modified polystyrene, 2 m diameter) labelled with a fluorescent yellow-green fluorochrome (fluorochrome detectable in the FITC channel) (Sigma-Aldrich, St. Louis/MO, USA) for 5 beads/cell and 10 beads/cell conditions, respectively. The wells were incubated in darkness at 37 C. in 5% CO.sub.2 atmosphere for 4 hours.

(31) After this incubation period, cells from each well were collected in a tube using phosphate buffer saline solution (PBS) (pH 7.4). (Note: after the incubation period, supernatants can be saved for further analysis by ELISA or CBA approaches, for instance). If necessary, a cell scraper was used to detach the adherent cells. Then, cells were centrifuged at 400 g for 10 minutes. The supernatant was discarded and the cell pellet was resuspended in 2 mL of PBS. Another centrifugation (400 g, 10 min) was performed and cells were finally resuspended in 0.1 mL of PBS solution.

(32) Afterwards, each sample was incubated with five monoclonal antibodies. The specificity of the monoclonal antibodies used, their origins and their fluorochromes were as follows: CD3 APC-H7 (Clone SK7); T-lymphocyte marker (BD, San Jose/Calif., USA) CD4 PE (Clone HP2/6); cytotoxic T-lymphocyte marker (ImmunoStep, Salamanca) CD14 PE-Cy7 (Clone RM052); monocyte marker (BD, San Jose/Calif., USA) CD45 PerCPCy5.5 (Clones 2D1); pan-leucocyte marker (Becton Dickinson Biosciences, BD, San Jose/Calif., USA) TNFalpha APC (Clone MAb11); immune response marker (BD, San Jose/Calif., USA)

(33) The tubes were gently vortexed and incubated in the dark for 15 minutes at room temperature. Immediately after this incubation period, 2 mL of PBS was added to each tube followed by gentle vortex. Then, samples were centrifuged at 400 g for 5 minutes. The supernatant was discarded and a new centrifugation step was performed. Afterwards, 0.2 mL of 1 annexin V binding buffer was added followed by the annexin V-CFBlue apoptosis marker (ImmunoStep, Salamanca/Spain). Cells were incubated for 15 minutes at room temperature and were stored at 4 C. in the darkness until analyzed in the flow cytometer.

(34) Note: samples from Example 1 can be employed as controls for activation of the immune response as well as for cell apoptosis.

(35) 2.3. Data Acquisition and Analysis

(36) Data acquisition was performed on a FACSCanto II flow cytometer (Becton Dickinson Biosciences, BD, San Jose/Calif., USA) using the FACSDiva software (v6.1; BD). For data analysis, the Infinicyt software (Cytognos SL, Salamanca, Spain) was used.

(37) Data analysis was performed in five steps. First, we selected the cells of interest (white blood cells) and excluded cell debris and cell aggregates, based on the expression of CD45 and light dispersion characteristics (forwards side scatterFSCand sideward side scatterSSC). Secondly, we identified and selected the monocytes (CD14.sup.++/CD4.sup.+/CD3.sup./CD45.sup.+), the T-CD4.sup.+ lymphocytes (CD45.sup.++/CD3.sup.+/CD4.sup.+/CD14.sup.), and the T-CD4.sup. lymphocytes (CD45.sup.++/CD3.sup.+/CD4.sup./CD14.sup.) (FIG. 3). Thirdly, we selected Annexin V-CFBlue positive cells within each population corresponding to apoptotic cells (FIG. 4). Afterwards, non-annexin positive cells (corresponding to viable cells) were used for determining TNFalpha-APC positive cells (for measuring the immune response) (FIG. 5). The effect of the stimulation in each cell population was assessed on unstimulated samples as a control and TNFalpha production was based on the percentage of positive cells, after subtracting the percentage of cells staining above the threshold for positivity in the negative control. Finally, phagocytic cells were identified selecting positive cells in the FITC channel (FIG. 6).

(38) 2.4. Results

(39) PBMN cells incubation with carboxylate-modified polystyrene 2 m fluorescent yellow-green latex beads did not induce apoptosis in any of the T cell populations analyzed (less than 1% apoptosis induction compared to a control without beads) at both 5 and 10 beads/cell ratio. Conversely, slight induction of apoptosis was detected on monocytes (18.73.2% and 21.80.3% Annexin V.sup.+ cells with a ratio of 5 and 10 beads/cell, respectively, vs. 13.13.7% in the control without beads).

(40) Similarly, incubation with latex beads did not lead to activation of either monocytes or T cells (less than 5% TNFalpha.sup.+ cells vs. baseline activation detected in the control condition without beads) at both ratios tested.

(41) Phagocytosis of latex beads was significantly observed on monocytes, with higher phagocytosis levels for conditions incubated at a ratio of 10 beads/cell compared to 5 beads/cell (72.42.0% vs. 48.70.7%, respectively).

(42) 2.5 Conclusion

(43) According to these results, this novel approach allows in one single step the simultaneous multiparametric and quantitative analysis of phagocytosis, viability and immune response against dye-labeled beads.

Example 3Assessment of Phagocytosis Using Non-Fluorescent Microparticles

(44) 3.1. Material and Methods

(45) Peripheral blood (PB) was obtained by venous puncture from 2 healthy individuals, placed in liquid lithium heparin tube and processed within the first 2 hours after collection for simultaneous analysis of viability, phagocytosis and immune response to incubation with microcapsules.

(46) 3.2. Sample Preparation

(47) Peripheral blood mononuclear cells (PBMN cells) were purified by density gradient centrifugation using Biocoll Separating Solution (Biochrom, Germany), according to the manufacturer's protocol. After centrifugation, the PBMN cell interphase was collected and quantified using a Neubauer chamber.

(48) A total of 0.210.sup.6 PBMCs were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin and 40 M TACE inhibitorTAPI-2(Sigma-Aldrich, St. Louis/MO, USA) in a 24-well plate (2 cm.sup.2 diameter). TAPI-2 was used as secretion blocking agent to reveal the levels of TNFalpha. The sample was incubated with 110.sup.6 and 210.sup.6 non-fluorescent beads (2 m diameter) for 5 beads/cell and 10 beads/cell conditions, respectively. The wells were incubated in darkness at 3 C. in 5% CO.sub.2 atmosphere for 4 hours.

(49) After this incubation period, cells from each well were collected in a tube using phosphate buffer saline solution (PBS). If necessary, a cell scraper was used to detach the adherent cells. Then, cells were centrifuged at 400 g for 10 minutes. The supernatant was discarded and the cell pellet was resuspended in 2 mL of PBS. Another centrifugation (400 g, 10 minutes) was performed and cells were finally resuspended in 0.1 mL of PBS solution.

(50) Afterwards, each sample was incubated with five monoclonal antibodies. The specificity of the monoclonal antibodies used, their origins and their fluorochromes were as follows: CD3 APC-H7 (Clone SK7); T-lymphocyte marker (BD, San Jose/Calif., USA) CD4 PE (Clone HP2/6); cytotoxic T-lymphocyte marker (ImmunoStep, Salamanca) CD14 PE-Cy7 (Clone RM052); monocyte marker (BD, San Jose/Calif., USA) CD45 PerCPCy5.5 (Clones 2D1); pan-leucocyte marker (BD, San Jose/Calif., USA) TNFalpha APC (Clone MAb11); immune response marker (BD, San Jose/Calif., USA)

(51) The tubes were gently vortexed and incubated in the dark for 15 minutes at room temperature. Immediately after this incubation period, 2 mL of PBS was added to each tube followed by gentle vortex. Then, samples were centrifuged at 400 g for 5 minutes. The supernatant was discarded and a new centrifugation step was performed. Afterwards, 0.2 mL of 1 annexin V binding buffer was added followed by the annexin V-CFBlue apoptosis marker (ImmunoStep, Salamanca/Spain). Cells were incubated for 15 minutes at room temperature and were stored at 4 C. in the darkness until analyzed in the flow cytometer.

(52) 3.3. Data Acquisition and Analysis

(53) Data acquisition was performed on a FACSCanto II flow cytometer (Becton Dickinson Biosciences, BD, San Jose/Calif., USA) using the FACSDiva software (v6.1; BD). For data analysis, the Infinicyt software (Cytognos SL, Salamanca, Spain) was used.

(54) Data analysis was performed in five steps. First, we selected the cells of interest (white blood cells) and excluded cell debris and cell aggregates, based on the expression of CD45 and light dispersion characteristics (forwards side scatterFSCand sideward side scatterSSC). Secondly, we identified and selected the monocytes (CD14.sup.++/CD4.sup.+/CD3.sup./CD45.sup.+), the T-CD4.sup.+ lymphocytes (CD45.sup.++/CD3.sup.+/CD4.sup.+/CD14.sup.), and the T-CD4.sup. lymphocytes (CD45.sup.++/CD3.sup.+/CD4.sup./CD14.sup.) (FIG. 3). Thirdly, we selected Annexin V-CFBlue positive cells within each population corresponding to apoptotic cells (FIG. 4). Afterwards, non-annexin positive cells (corresponding to living cells) were used for determining TNFalpha-APC positive cells (for measuring the immune response) (FIG. 5). The effect of the stimulation in each cell population was assessed on unstimulated samples as a control and TNFalpha production was based on the percentage of positive cells, after subtracting the percentage of cells staining above the threshold for positivity in the negative control. Finally, phagocytic cells were identified by comparing the SSC/FSC plot obtained with size-matched fluorescent-beads (Example 2). After selecting phagocytic cells as FITC positive cells (for fluorescent beads), SSC/FSC plot was employed as a template for phagocytic cells of non-fluorescent beads (FIG. 7).

(55) 3.4 Results

(56) Analysis strategy for phagocytosis assessment of fluorescent beads was performed in the same samples using the methodology for analysis of phagocytosis of non-fluorescent beads. Overall, a significant correlation was observed between the two methods of analysis (R.sup.2=0.934; p<0.01), as similar results were obtained for both the 5 beads/cell (57.00.8% phagocytosis using the method for fluorescent beads vs. 52.54.5% phagocytosis using the method for non-fluorescent beads) and 10 beads/cell (77.32.8% phagocytosis using the method for fluorescent beads vs. 75.62.6% phagocytosis using the method for non-fluorescent beads) conditions.