The aluminium adjuvant preparations used herein were Alhydrogel^®, AlO(OH), and Adju-Phos^®, Al(OH)x(PO₄)y, purchased from Brenntag Biosector (Fredikssund, Denmark). Zeolite Y (NaY) and dealuminated zeolite Y (USY) were manufactured by Tosoh cooperation, Japan and before use, the zeolites were sterilized by heating for 3 hours at 300 °C and fractionated as earlier described [8Ohlsson L, Exley C, Darabi A, Sandén E, Siesjö P, Eriksson H. Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells. J Inorg Biochem 2013; 128: 229-36.
[http://dx.doi.org/10.1016/j.jinorgbio.2013.08.003] [PMID: 23992993] ].

Lumogallion (CAS 4386-25-8) came from TCI Europe N.V., Antwerp, Belgium and thiazolyl blue tetrazolium bromide (MTT), M2128 was purchased from Sigma-Aldrich, St. Louis, MO, USA.

THP-1, (ATCC TIB-202), was obtained from LGC Standards, UK and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum of EU grade, (Gibco, ThermoFisher Scientific), and 100 μg/ml of gentamicin (Corning Media Tech, ThermoFisher Scientific). This medium will be referred to as R10. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂ and the cells were maintained by sub-culturing once every third day.

Alhydrogel^®, AlO(OH), and Adju-Phos^®, Al(OH)x(PO₄)y, corresponding to 4 mg aluminium/ml were incubated over night at room temperature on a rocking table with lumogallion, 0 – 100 μM, in a total volume of 1 ml 150 mM NaCl. The next day the adjuvants were collected by centrifugation 10 minutes at 13,000 x g. The adjuvants were washed with 1 ml 150 mM NaCl and finally carefully re-suspended using a pipette, in 150 mM NaCl to a concentration of 4 mg aluminium per ml [7Mile I, Svensson A, Darabi A, Mold M, Siesjö P, Eriksson H. Al adjuvants can be tracked in viable cells by lumogallion staining. J Immunol Methods 2015; 422: 87-94.
[http://dx.doi.org/10.1016/j.jim.2015.04.008] [PMID: 25896212] ]. Fluorescence of the labelled ABA preparations was measured by adding 100 μl in triplicates of each of the re-suspended preparations in a 96 well plate and measured using a SpectraMAX GEMINI XS, Molecular Devices, with an excitation wave length of 490 nm, emission wave length of 580 nm, and a cut of filter of 510 nm.

After labelling and washing Alhydrogel^® and Adju-Phos^® as described, the labelled adjuvant preparations were re-suspended in 150 mM NaCl or R10 culture medium to a concentration of 4 mg/ml. Re-suspended adjuvants were sonicated in an ultrasonic bath for 5 min and diluted with Milli-Q water to a concentration of 20 μg/ml. The Zeta potential of the diluted suspensions was determined using a NICOMP 380 ZLS Electrophoretic Light Scattering instrument from Particle Sizing Systems (Port Richey, FL, USA). The temperature was adjusted to 23 °C during measurements and determinations were done in phase analysis mode. The dielectric constant (78.5), refractive index (1.33) and viscosity (0.93 mPas) of pure water were used for calculations. The size distribution of the adjuvant preparations was determined using a Coulter LS230 Laser Diffraction Particle Size Analyser equipped with a Small Volume Module (Beckman Coulter, CA, USA).

Size determination by flow cytometry: THP-1 cells, 1.4 x 10⁶ cells/ml, were mixed with an equal volume of R10 cell culture medium, Alhydrogel^® 200 μg/ml in R10 culture medium or Adju-Phos^® 200 μg/ml in R10 culture medium. After one hour at 37 °C, the samples were analysed and the cell concentration in the samples was calculated from events collected in the cell gate (P3 shown in Fig. 3).

Leukocytes were isolated by lysis of red blood cells by ammonium chloride. Briefly, 6 ml of the blood from healthy donors were suspended in 54 ml cold lysis solution containing 8% (w/v) NH₄Cl in 10 mM sodium bicarbonate and 1 mM EDTA. The cell suspension was incubated for 10 min on ice, collected by centrifugation for 5 min at 500 x g, washed two times, each time with 10 ml R10 culture medium and finally re-suspended in R10 medium at a concentration of 2 x 10⁶ cells per ml and stored on ice until use.

Leukocytes at a final concentration of 1 x 10⁶ cells per ml were co-cultured 2 h at 37 °C in a 24 well plate with Alhydrogel^® pre-labelled with 100 μM lumogallion, or with non-labelled ABA as control, at a concentration of 12.5 μg or 50 μg aluminium/ml in a total volume of 1200 μl R10 culture medium. In a second experimental set up leukocytes,1 x 10⁶ cells per ml, were co-cultured with non-labelled Alhydrogel^® or Adju-Phos^® at final concentrations of 200, 400 and 800 μg aluminium/ml. In this experimental set up, controls were composed of cells cultured in plain culture medium (R10).

The cells were harvested from the plate and each of the incubations was divided into 6 aliquots of 200 μl and stained on ice for 30 min with APC labelled antibodies against CD3, CD11b, CD14, CD19 or an isotype control (all antibodies from BD Bioscience, San Jose, CA, USA, except the isotype control that came from R&D Systems, Minneapolis, MN, USA). The stained cells were then diluted with 500 μl cold PBS containing 0.1% (w/v) BSA and were direct, without any washing, analysed using an Accuri C6 flow cytometer and standard settings. From each sample, 20 000 leukocytes were collected and the different leukocyte populations were identified by their FSC x SSC scatter. Gate settings (FSC x SSC) were done on the various leukocyte populations (lymphocytes, monocytes and granulocytes) and their association with aluminium adjuvants was examined by their lumogallion fluorescence. In experiments using 200-800 μg aluminium/ml of non-labelled ABAs, the leukocyte populations were identified by gating antibody positive events into I give up an FSC x SSC dot plot and the number of events in the viable FSC x SSC gate of the corresponding leukocyte population was measured. Leukocytes from three donors were isolated and studied.

THP-1 cells, 1 x 10⁶ cells/ml, were incubated for 20 min at 37 °C in PBS containing 0.25 μM CellTrace™ Far Red (Life Technologies, ThermoFisher Scientific, MA USA). R10 culture medium was added to the incubation and the cells were collected by centrifugation for 10 min at 300 x g. The cells were re-suspended in R10 medium at a cell concentration of 0.8 x 10⁶ cells/ml and incubated over night at 37 °C with Alhydrogel^® or Adju-Phos^®, pre-labelled with 100 μM lumogallion, at a concentration of 100 μg aluminium/ml, in a total volume of 5ml R10 medium in T25 culture flasks. Controls were composed of unstained or CellTrace™ stained THP-1 cells incubated in plain culture medium (R10).

The next day the cells were collected by centrifugation for 10 min at 300 x g and re-suspended in 1 ml R10 medium. Cell suspensions, 1 ml, were separated from free adjuvant by density centrifugation on 5 ml Ficoll-Paque^TM (GE Healthcare Life Sciences, Uppsala, Sweden) for 25 min at 600 x g. Cells at the interphase were isolated, washed with PBS and re-suspended in R10 culture medium at 0.13 x 10⁶ cells/ml. 200 μl of the cell suspension was transferred to the wells in a 96 well plate and then sub-cultured at 37 °C. At the start of the sub-culture and after 24, 48 and 72 h of sub-culture, cells from four wells were collected and their fluorescence was analysed using an Accuri C6 flow cytometer and standard settings.

Sub-cultured THP-1 cells containing intracellular lumogallion labelled aluminium adjuvants and stained with CellTrace™ Far Red were withdrawn and collected by centrifugation, 5 minutes at 1,000 x g. The cells were re-suspended in 1% (w/v) PFA, incubated for 15 minutes at room temperature, collected by centrifugation for 5 minutes at 1,000 x g and washed twice with PBS. Finally, the cells were re-suspended in a small volume of PBS and mounted on microscope slides using ProLong^® Gold Antifade Mountant with DAPI (Life Technologies, ThermoFisher Scientific, MA USA). After mounting, the samples were analysed on a Zeiss LSM 780 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). DAPI was excited with the 405 nm laser and emitted light was detected between 410-493 nm. Lumogallion was excited with the 488 nm laser and emitted light was collected between 534-607 nm. CellTrace™ Far Red dye was excited with the 633 nm laser and emitted light was collected between 650-743 nm. Z-stack images were obtained at 63x magnification and analysed with ZEN 2012 (Carl Zeiss Microscopy GmbH).

Two sets of triplicates of THP-1 cells 0.13 x 10⁶ cells per ml, were co-cultured in 96 well plates with Alhydrogel^®, Adju-Phos^®, zeolite Y (NaY) or dealuminated zeolite Y (USY) in a total volume of 200 μl R10 culture medium for 1 to 2 days at 37 °C. Co-cultures with aluminium adjuvants were done at final aluminium concentrations ranging from 6.26 to 100 μg/ml. NaY was used at concentrations ranging from 3.8 to 30 μg/ml whereas USY was used at concentrations ranging from 1.25 to 10 μg/ml. Prior to use, the zeolites were size fractionated and quantified using light scattering as previously described [9Dahm A, Eriksson H. Ultra-stable zeolites--a tool for in-cell chemistry. J Biotechnol 2004; 111(3): 279-90.
[http://dx.doi.org/10.1016/j.jbiotec.2004.04.008] [PMID: 15246664] ].

Controls consisted of cells cultured in R10 medium without any further additions. For the MTT assay, corresponding concentrations of aluminium adjuvants and zeolites in cell free R10 medium were assayed.

After co-culture of cells and particles (ABAs or zeolites) for 1 respectively 2 days, one set of triplicates from each of the particle concentrations was pooled and the number of cells was analysed using an Accuri C6 flow cytometer with an FSC x SSC live cell gate collecting 10,000 events.

MTT assays were performed on the other set of triplicates, and the controls, vide supra [8Ohlsson L, Exley C, Darabi A, Sandén E, Siesjö P, Eriksson H. Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells. J Inorg Biochem 2013; 128: 229-36.
[http://dx.doi.org/10.1016/j.jinorgbio.2013.08.003] [PMID: 23992993] , 10Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65(1-2): 55-63.
[http://dx.doi.org/10.1016/0022-1759(83)90303-4] [PMID: 6606682] ]. Briefly 25 μl of the MTT reagent (5 mg/ml in PBS) was added to each well. The samples were incubated for another 2 h at 37 °C. After 2 h, 60 μL lysis buffer was added to solubilize the MTT-formazan product and the cells were incubated for another 24 h before the absorbance at 540 nm was read using a microplate reader.

The control, obtained after addition of the MTT reagent to cell free triplicates of particles in culture medium was subtracted from the absorbance obtained after co-culture with THP-1 cells.

Based on the estimated number of cells in the samples (by flow cytometry) and the MTT activity (absorbance) from the samples, the MTT activity was expressed as MTT activity per 10⁶ cells. The MTT activities per 10⁶ cells were then normalized against the MTT-activity per 10⁶ cells obtained in plain culture medium at the appropriate day of the experiments. Three independent experiments were performed and the results are presented as mean +/- SD.

MACS technology based on magnetically labelling cells and retaining cells on a column was used to isolate monocytes (Monocyte isolation kit II, Miltenyi Biotec, Bergisch Gladbach, Germany).

Briefly, peripheral blood mononuclear cells (PBMC) were obtained from human leucocyte concentrates (Lund University Hospital, Sweden) from healthy donors by density centrifugation on Ficoll-Paque^TM (GE Healthcare Life Sciences, Uppsala, Sweden). Untouched CD14⁺ monocytes were isolated by indirect magnetic labelling of non-monocytes with a cocktail of biotin-conjugated antibodies against CD3, CD7, CD16, CD19, CD56, CD123 and CD235a followed by the addition of anti-Biotin MicroBeads. Non CD14⁺ monocytes were depleted on a MACS column and cells in the flow through were collected, washed and re-suspended in R10 medium at 1 x 10⁶ cells per ml. The purity of the monocytes used in the experiments was 85-92% based on the binding of a FITC labelled anti CD14 antibody (BD Bioscience, San Jose, CA, USA) and flow cytometry.

Triplicates of isolated peripheral monocytes, final concentration 0.5 x 10⁶ cells per ml, were incubated in 96 well plates with Alhydrogel^® or Adju-Phos^® corresponding to final aluminium concentrations ranging from 6.3 to 100 μg/ml in a total volume of 200 μl R10 culture medium during 1h or 4h at 37 °C.

One control consisted of cells cultured in R10 medium without any further additions and another control consisted of corresponding concentrations of ABA in cell free R10 medium.

After 1 respectively 4 h the MTT assay was performed by adding 25 μl of MTT reagent to each well as previously described. Based on the added number of cells in the samples (100.000 cells per well) and the MTT activity (absorbance) from the samples, the MTT activity was expressed as MTT activity per 10⁶ cells. The MTT activities were then normalized against the MTT-activity obtained in plain culture medium of the 1 respective 4 h experiments as previously described. Three independent experiments were performed using different blood donors and the results are presented as mean +/- SD.

Nutrient depleted medium was prepared by supplementing a solution of 65.6 mM NaCl, 3.2 mM KCl, 0.7 mM MgSO₄, 1.6 mM CaCl₂, 0.6 M Na₂HPO₄, 23.9 mM NaHCO₃ and 6.3 mM Na-citrate, pH 7,4 with 10 mM Hepes (Life Technologies, ThermoFisher Scientific, MA USA), 3.6 mM glucose, 2 mM Glutamax (Life Technologies, ThermoFisher Scientific, MA USA), 1x non-essential amino acids (Life Technologies, ThermoFisher Scientific, MA USA), 1x MEM vitamin solution (Sigma-Aldrich, St. Louis, MO, USA), 10% foetal calf serum of EU grade, and 100 μg/ml of gentamicin.

Two sets of triplicates of THP-1 cells 0.13 x 10⁶ cells per ml, were cultured in 96 well plates in a total volume of 200 μl nutrient depleted medium or in R10 medium for 1 to 2 days at 37 °C. Controls consisted of an extra set of triplicates of THP-1 cells cultured in R10 medium.

After co-culture of cells and particles (ABAs or zeolites) for 1 respectively 2 days, one set of triplicates from each of the particle concentrations was pooled and the number of cells was analysed using an Accuri C6 flow cytometer with an FSC x SSC live cell gate collecting 10,000 events. The other set of triplicates and the controls containing nutrient depleted medium or cell free R10 medium, were used for the MTT assay, as previously described.

Based on the estimated number of cells in the samples (by flow cytometry) and the MTT activity (absorbance) from the samples, the MTT activity was expressed as MTT activity per 10⁶ cells. The MTT activities per 10⁶ cells were then normalized against the MTT-activity per 10⁶ cells obtained in the extra set of triplicates of THP-1 cells cultured in R10 medium at the appropriate day of the experiments

Three independent experiments were performed and the results are presented as mean +/- SD.

Incubation of ABAs with lumogallion, a fluorimetric reagent for aluminium ions [6Mold M, Eriksson H, Siesjö P, Darabi A, Shardlow E, Exley C. Unequivocal identification of intracellular aluminium adjuvant in a monocytic THP-1 cell line. Sci Rep 2014; 4: 6287.
[http://dx.doi.org/10.1038/srep06287] [PMID: 25190321] ], creates highly fluorescent labelled ABA preparations [7Mile I, Svensson A, Darabi A, Mold M, Siesjö P, Eriksson H. Al adjuvants can be tracked in viable cells by lumogallion staining. J Immunol Methods 2015; 422: 87-94.
[http://dx.doi.org/10.1016/j.jim.2015.04.008] [PMID: 25896212] ]. Of the two aluminium salts most commonly used as adjuvants, aluminium oxyhydroxide, (Alhydrogel^®), shows a linear increase in fluorescence upon incubation with lumogallion, whereas aluminium hydroxyphosphate, (Adju-Phos^®) becomes saturated (Fig. 1).

Fig. (1) Fluorescence labelling of aluminium adjuvants. Aluminium adjuvants were labelled with various concentrations of lumogallion by overnight incubation, washed and re-suspended in 150 mM NaCl. Fluorescence from re-suspended Alhydrogel® (●) and Adju-Phos® (■) particles was measured by excitation at 490 nm and emission at 580 nm.

Physicochemical characterisation of the labelled ABAs showed no or minor effects on the zeta potential after re-suspension in NaCl and a slightly decreased zeta potential after labelling Adju-Phos^® with the highest investigated concentrations of lumogallion (50 and 100 μM) and re-suspension into culture medium containing 10% serum (Table 1).

Table 1
Zeta potential after labelling aluminium adjuvants with lumogallion.
Aluminium adjuvants were labelled with various concentrations of lumogallion and after washing suspended at a concentration of 4 mg/ml in either 150 mM NaCl or cell culture medium, R10. Number of zeta potential determinations (n) was 6.

No significant change of size distribution was observed due to lumogallion labelling, however, re-suspension of both Alhydrogel^® and lumogallion labelled Alhydrogel^® in culture medium containing 10% serum, resulted in a significantly increased size distribution of the adjuvant (Fig. 2).

Fig. (2) Size distribution of aluminium adjuvant particles. Aluminium adjuvants, non-labelled and lumogallion labelled, were re-suspended in 150 mM NaCl or cell culture medium containing 10% fetal calf serum (R10) before size determination of the adjuvant particles. A. Alhydrogel® re-suspended in 150 mM NaCl (❍) or in cell culture medium (●). B. Adju-Phos® re-suspended in 150 mM NaCl (❏) or in cell culture medium (■). C. Lumogallion (100 μM) labelled Alhydrogel® re-suspended in 150 mM NaCl (❍) or in cell culture medium (●). D. Lumogallion (100 μM) labelled Adju-Phos® re-suspended in 150 mM NaCl (❏) or in cell culture medium (■).

The size distribution of Alhydrogel^® showed surprisingly large particles after resuspension in culture medium containing serum and this was quite different from the particle size estimated by forward scatter versus side scatter using flow cytometry (Fig. 3), or visualized by light microscopy, in which the Alhydrogel^® particles are clearly smaller than THP-1 cells which have an approximate size of 10-15 μm.

Flow cytometry detects cells and particles by their forward and side scatter and by a gate setting, P3, as outlined in (Fig. 3), information regarding THP-1 cells, such as a number of events and sample volume can be collected and used to calculate the number of cells/ml in the sample. The presence of 16 μm and larger ABA particles, as shown by laser diffraction of Alhydrogel^® suspended in R10 medium (Fig. 2A), would be detected in the same gate as THP-1 cells, but no such particles were detected in the THP-1 gate (Fig. 3). Neither could any particles of this size be observed in light microscope and after incubation of 1.4 x 10⁶ cells per ml with an equal volume of culture medium, Alhydrogel^® or Adju-Phos^® suspensions, an expected 50% reduction of the cell concentration, i.e. 0.7 x 10⁶ cells per ml, was obtained in all incubation mixtures using a gate setting on the THP-1 cells, as P3 shown in (Fig. 3) to calculate the cell concentration.

Fig. (3) Flow cytometry of lumogallion labelled aluminium adjuvants and THP-1. Lumogallion labelled ABAs suspended in R10 medium were incubated with and without THP-1 cells for 1 h at 37°C and then analysed by flow cytometry using forward (FSC) and side (SSC) scatter. Gate setting used to analyse THP-1 cells (P3) and ABA particles (P4). A. Lumogallion labelled Alhydrogel®, 100 μg/ml in R10 medium. B. Lumogallion labelled Adju-Phos®, 100 μg/ml in R10 medium. C. THP-1 cells incubated with R10 medium. D. THP-1 cells incubated with Alhydrogel®, 100 μg/ml in R10 medium. E. THP-1 cells incubated with Adju-Phos®, 100 μg/ml in R10 medium.

Lumogallion labelled ABAs can be utilized to investigate the interaction between the adjuvants and different leukocyte populations in peripheral blood. By flow cytometry, the various leukocyte populations can be identified and their association with the different types of ABA can be explored (Fig. 4).

Fig. (4) shows cells associated with ABAs in the upper right quadrant after co-culture with a low concentration of ABA (12.5 μg/ml) whereas a summary of leukocyte association with the two main forms of ABA after co-culture with a four times higher concentration of ABAs (50 μg/ml) is presented in (Table 2). Based on experiments with leukocytes from three independent donors (Table 2), a more pronounced cellular association was observed for aluminium oxyhydroxide (Alhydrogel), with B-lymphocytes being the exception.

Cellular association with ABA particles, either adsorbed on the cell surface or endocytosed, may affect the condition and viability of the cells. Further increasing the ABA concentration from 100 to 800 μg/ml showed almost no effect on the recovered and living number of granulocytes whereas decreased recovery was observed for both monocytes and lymphocytes (Fig. 5). A clear difference regarding cell recovery between the two forms of ABA was also observed, showing a much lower recovery after incubation with Adju-Phos^® (Fig. 5) compared to Alhydrogel^®.

Table 2
Al adjuvant associated with human peripheral leukocytes.
Leukocytes were incubated with 50 μg/ml of lumogallion labelled ABAs and the percentage fluorescent cells of each population, i.e. percentage of lymphocytes, monocytes or granulocytes associated with ABAs, was determined. Results based on leukocytes isolated from three different donors.

Fig. (4) Cellular association of aluminium adjuvant determined by flow cytometry. Human peripheral leucocytes were incubated with a low concentration (12.5 μg/ml) of lumogallion labelled Alhydrogel® or with non-labelled Alhydrogel® before the cells were stained with fluorescently labelled antibodies against T-lymphocytes, monocytes and granulocytes. Lymphocytes, monocytes and granulocytes were identified by FSC x SSC dot plots and after gate settings on respective populations, their association with fluorescent ABAs was determined. A-C; Cells incubated in the presence of non-labelled Alhydrogel®. D-F; Cells incubated in the presence of lumogallion labelled Alhydrogel®. A and D; T-lymphocytes identified by antibodies against CD3. B and E; Monocytes identified by antibodies against CD14. C and F; Granulocytes identified by antibodies against CD11b.

ABAs probed with lumogallion have earlier been shown to be endocytosed by cells [7Mile I, Svensson A, Darabi A, Mold M, Siesjö P, Eriksson H. Al adjuvants can be tracked in viable cells by lumogallion staining. J Immunol Methods 2015; 422: 87-94.
[http://dx.doi.org/10.1016/j.jim.2015.04.008] [PMID: 25896212] ] and labelling cells with CellTrace™ Far Red makes it possible to trace multiple generations by flow cytometry and dye dilution (Fig. 6A). Intracellular ABAs labelled with lumogallion can clearly be visualized inside cells co-stained with the cytoplasmic stain CellTrace™ Far Red using confocal microscopy (Fig. 6B) and by following the dilution of the CellTrace dye and at the same time also follow the reduction of fluorescence from endocytosed lumogallion labelled ABAs, the elimination of adjuvants from the cells can be investigated.

Fig. (5) Recovered human leukocytes after incubation with various concentrations of adjuvants. Human leukocytes were co-cultured for 2h with different concentrations of Alhydrogel® (black pile) respective Adju-Phos® (grey pile). By means of specific CD-markers four different populations of leukocytes were identified using flow cytometry and cells of the respective leukocyte population are expressed as % of cells obtained after incubation in culture medium. A. T-lymphocytes. B. Monocytes. C. Granulocytes and D. B-lymphocytes. Data presented as mean +/- SD of three independent donors.

Fig. (6A) Flow cytometry of cells stained with CellTrace™ Far Red immediately after incubation with aluminium adjuvants. THP-1 cells stained with the cytoplasm stain CellTrace™ Far Red were incubated with lumogallion labelled ABAs. The cells were separated from free adjuvant particles and re-suspended in culture medium. Experimental conditions as described in the Materials and Methods section. Left histogram: Aluminium adjuvant fluorescence intensity (lumogallion) associated with THP-1 cells immediately after separation from free ABAs in the culture medium. Black histogram: Cells incubated with lumogallion labelled Alhydrogel®. Grey histogram: Cells incubated with lumogallion labelled Adju-Phos®. White histogram: Cells incubated in culture medium. Right histogram: Cytoplasm fluorescence intensity (CellTrace™ Far Red) from THP-1 cells immediately after separation from free ABAs in the culture medium. Black histogram: Cells incubated with lumogallion labelled Alhydrogel®. Grey histogram: Cells incubated with lumogallion labelled Adju-Phos®. White histogram: Non-stained cells in culture medium. Histograms of cells stained with CellTrace™ Far Red and incubated with the two forms of lumogallion labelled ABAs showed the same fluorescence intensity (black and grey histograms are completely overlapping).

Fig. (6B) Visualization of intracellular aluminium adjuvant. Confocal images of the centre sections of THP-1 cells that after staining with CellTrace™ Far Red were incubated with lumogallion labelled ABAs, washed and put back in culture. Experimental conditions as described in the Materials and Methods section. Images show nucleus as blue (DAPI), cytoplasm as red (CellTrace™ Far Red) and aluminium adjuvant as green (lumogallion). Upper left. A cell immediately after incubation with lumogallion labelled Alhydrogel® and removal of non-phagocytosed ABA. Upper right. A cell 3 days in culture after incubation with lumogallion labelled Alhydrogel® and removal of non-phagocytosed ABA. Lower left. A cell immediately after incubation with lumogallion labelled Adju-Phos® and removal of non-phagocytosed ABA. Lower right. A cell without any incubation with aluminium adjuvants.

However, extracellular, not endocytosed ABA particles, must be removed before commencing the fluorescence measurements. To obtain THP-1 cells with only endocytosed and intra cellular ABAs, extracellular ABAs were removed by density centrifugation, allowing the collection of THP-1 cells in the interphase, whereas ABA particles are centrifuged through the density barrier. (Figs. 6A and 6B) show the fluorescence of CellTrace™ Far Red stained THP-1 cells containing intracellular lumogallion labelled ABAs at day zero before sub-culturing of the cells begins. Cells were then sub-cultured at 37°C in a humidified atmosphere with 5% CO₂. Samples were withdrawn every 24 hours and the cellular fluorescence was examined by flow cytometry and expressed as per cent of the fluorescence intensity obtained at day zero (Fig. 6C).

Fig. (6C) Mean fluorescence intensity from stained THP-1 cells in culture. Cytoplasm stained THP-1 cells continuously cultured after separation from lumogallion labelled adjuvants in the culture medium. At various days, samples were withdrawn, analysed by flow cytometry and the mean fluorescence intensity (MFI) was expressed as % of MFI obtained immediately after separation from free ABAs in the culture medium (Day 0). Experimental conditions as described in the Materials and Methods section. Left graph: Cells incubated with Alhydrogel®; lumogallion fluorescence (■) and CellTrace™ Far Red fluorescence (❍). Right graph: Cells incubated with Adju-Phos®; lumogallion fluorescence (■) and CellTrace™ Far Red fluorescence (❍).

The intracellular presence of ABA will undoubtedly affect the cells and in an earlier report, THP-1 cells showed reduced proliferation on co-culture with ABAs [8Ohlsson L, Exley C, Darabi A, Sandén E, Siesjö P, Eriksson H. Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells. J Inorg Biochem 2013; 128: 229-36.
[http://dx.doi.org/10.1016/j.jinorgbio.2013.08.003] [PMID: 23992993] ]. Reduced proliferation was observed after co-culture of THP-1 cells with the two forms of ABA, as well as after co-culture with zeolite Y (NaY) and with dealuminated zeolite Y (USY). Both Alhydrogel^® and USY decreased the cell growth by approximately 13-15% at day 1, and by 25-30% at day 2 (Figs. 7A and 7C). The decrease owing to Adju-Phos^® and NaY was approximately the same, 10-15 % at day 1, and 20-30% at day 2.

Fig. (7) MTT activity after co-culture with Alhydrogel® and USY. THP-1 cells were co-cultured with various concentrations of Alhydrogel® or dealuminated zeolite Y (USY). At day 1 (■) and day 2 (▲) the number of cells in each of the incubations was counted and an MTT assay was performed. The MTT activity (absorbance) per 106 cells was calculated and to normalize the samples, the ratio between MTT activity per cells in the various cell cultures and the results obtained from cells in culture medium without any further additions at the corresponding day was assessed. A. Cell number after culture with Alhydrogel®. Cell number expressed as % of cells obtained after culture in medium without any further additions. B. Normalized MTT activity ratio of THP-1 cells cultured in the presence of Alhydrogel®. Dotted line shows the expected normalized MTT ratio using the MTT assay to estimate the number of cells in a sample. C. Cell number after culture with USY. Cell number expressed as % of cells obtained after culture in medium without any further additions. D. Normalized MTT activity ratio of THP-1 cells cultured in the presence of USY. Data presented as mean +/- SD of three independent experiments.

The MTT assay is widely used to estimate the number of viable cells in a sample. The assay is based on the assumption that the quantity of formazan produced is directly proportional to the number of viable cells and that formazan production by all the individual cells in the sample is uniform [10Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65(1-2): 55-63.
[http://dx.doi.org/10.1016/0022-1759(83)90303-4] [PMID: 6606682] ]. However, during co-culture of THP-1 cells and ABAs, the MTT assay gives a misleading result showing a too high number of cells in the sample [8Ohlsson L, Exley C, Darabi A, Sandén E, Siesjö P, Eriksson H. Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells. J Inorg Biochem 2013; 128: 229-36.
[http://dx.doi.org/10.1016/j.jinorgbio.2013.08.003] [PMID: 23992993] ]. By calculating the MTT absorbance, i.e. the formazan production, per 10⁶ cells (number of viable cells identified by counting) in a sample, an escalating value was obtained upon co-culture with increasing concentrations of ABAs. To normalize the samples, the ratio between MTT-activity per 10⁶ cells and the MTT-activity per 10⁶ cells in plain culture medium was calculated for each separate day and in Figs. (7A and 7D) the results after co-culture in the presence of Alhydrogel^® and USY are shown.

All experiments were done in triplicates and the mean values with standard deviations are shown in (Table 3). The table presents values obtained from co-cultures with 10-15% reduced proliferation at day 1, and 20-30% at day 2. The results indicate an apparent increased normalized MTT activity ratio for both ABAs and the aluminium containing zeolite NaY, whereas the USY ratio was close to the expected ratio of one.

Table 3
Cellular MTT activity after culture of cells in the presence of Al-adjuvants, zeolites or depleted growth medium.
THP-1 cells were cultured in the presence of ABAs, zeolites or in depleted culture medium. At day 1 respectively day 2 the cell numbers were counted and an MTT assay of the various incubations was performed. The MTT activity (absorbance) per 1cells was calculated and to normalize the samples, the ratio between MTT-activity in plain culture medium was assessed for each separate day. Data presented as mean +/- SD of three independent experiments.

Co-culture with ABAs did not only affect the MTT activity of THP-1 cells but also affected human peripheral monocytes (Fig. 8). Co-culture for one hour (i.e. one-hour incubation with ABAs followed by two hours’ incubation with the MTT reagent) showed no significant increase of the normalized MTT activity, although a prolonged incubation with ABAs showed a significant increase of normalized MTT activity upon co-culture with Adju-Phos^®.

Fig. (8) MTT activity by human monocytes after co-culture with Al-adjuvants. Normalized MTT activity ratio of human peripheral monocytes cultured in the presence of Alhydrogel® (●) and Adju-Phos® (■). The MTT assay was performed either 1 h (A) or 4 h (B) after addition of ABAs to the monocytes. Data presented as mean +/- SD from three independent donors.

Finally, in an attempt to imitate the increased MTT activity per 10⁶ cells, THP-1 cells were cultured in nutrient depleted medium, resulting in a slightly increased normalized MTT activity (Table 3).