Aluminum ions (Al³⁺) bound to aluminum adjuvant particles/aggregates [2]

Lumogallion (1 mM, 45 minutes) can detect aluminum levels in the brain tissue of Alzheimer's sufferers [1]. Lumogallion can be used as a probe to stain live cells using an intracellular aluminum adjuvant [2]. Lumogallion (10 μM, 60 min) can detect aluminum levels in plant root tip cells [3]. Guidance (This is our recommended protocol. This procedure is intended for guidance and should be tailored to your individual requirements. 1. Dissolve the Lumogalion in 0.1 M acetate buffer (pH 5.2). 2. Incubate the sample with 10 μM lumogallion for 60 minutes in darkness. 3. Wash the mixed sample twice with acetate buffer for 15 minutes each, then examine it using a confocal laser microscope. 4. The excitation wavelength is 488 nm, while the emission wavelength is 520 nm.

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The compound exhibited strong fluorescence upon binding to intracellular aluminum adjuvants phagocytosed by viable cells. Fluorescence appeared as spots in the cytoplasm. Staining of living cells with lumogallion revealed internalization of aluminum adjuvant particles in both suspension and adherent cell lines (THP-1 and GL261). [2]
Pre-staining of aluminum adjuvants (Alhydrogel AlO(OH) and Adjub-Phos AlPO4) with lumogallion resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks, with only minor reduction of fluorescence upon extensive washing. Less than 20% of particle-associated fluorescence was dissipated during the second to tenth re-suspension/washing step. [2]
Lumogallion emission spectrum is extremely broad, ranging from 520 to 650 nm with a peak around 580 nm after excitation at 490 nm. The broad emission makes it difficult to combine with other fluorochromes emitting in the 520-620 nm range, but no interference is observed in the far-red region. [2]
Co-localization of lumogallion and LysoTracker Deep Red was clearly shown in GL261 cells co-cultured with aluminum adjuvant, indicating that aluminum adjuvants are localized in acidic vesicles (lysosomes). Not all lumogallion staining co-localized with LysoTracker, which occurs in early phagosomes where pH has not yet decreased. [2]
Compared to lumogallion, morin (another aluminum stain) gave fluorescence signals several magnitudes weaker when detecting intracellular aluminum adjuvants in viable cells. [2]

THP-1 cells (0.5×10⁶ cells/ml) were co-cultured with Alhydrogel AlO(OH) adjuvant (aluminum content 0.8 to 50 μg/ml) in R10 culture medium for 4 to 16 h at 37°C. Lumogallion was then added to final concentrations of 1.6 to 20 μM and cells were further incubated at 37°C for various time intervals (30 min up to overnight). After staining, cells were fixed in 1% (w/v) paraformaldehyde and analyzed by flow cytometry. [2]
THP-1 cells were co-cultured with lumogallion pre-stained Alhydrogel AlO(OH) (aluminum concentration 0.8 to 50 μg/ml) for 4 to 24 h at 37°C. Cells were then fixed and analyzed by flow cytometry. [2]
GL261 cells were seeded into culture chambers (1×10⁵ cells/ml) and allowed to adhere overnight. Aluminum adjuvant (0.3 to 10 μg/ml) was added and cells were incubated for 4 to 16 h. Lumogallion was added to 5 μM and incubated overnight. Cells were washed, fixed in 1% PFA, mounted with DAPI-containing mountant, and analyzed by confocal or fluorescence microscopy. [2]
For dual staining with FITC-labeled antibody: THP-1 cells were co-cultured with Alhydrogel (12.5 μg Al/ml) for 4 h, then stained with 5 μM lumogallion overnight. Cells were then stained with FITC-labeled anti-CD45 or isotype control (30 min on ice), washed, fixed, and analyzed by flow cytometry with 70% compensation for lumogallion overflow into green channel. [2]
For dual staining with MitoTracker Deep Red: THP-1 cells were co-cultured with lumogallion pre-stained Alhydrogel (12.5 μg Al/ml) overnight. Then 5 nM MitoTracker Deep Red was added for 30 min at 37°C. Cells were washed, fixed, and analyzed by flow cytometry without compensation. [2]
For dual staining with LysoTracker Deep Red: GL261 cells were co-cultured with 2.5 μg/ml AlO(OH) for 6 h, then 10 μM lumogallion was added overnight. Cells were then stained with 200 nM LysoTracker Deep Red for 1 h at 37°C, washed, fixed, mounted, and analyzed by confocal microscopy. [2]

No effect on viability of THP-1 cells was observed after incubation in the presence of 50 μM lumogallion for 24 h. Cell counts: in R10 medium, 0.91×10⁶±0.03×10⁶ cells/ml; after co-culture with 50 μM lumogallion, 0.86×10⁶±0.03×10⁶ cells/ml. MTT assay: absorbance at 600 nm was 1.07±0.04 in R10 medium and 1.14±0.04 in the presence of lumogallion. Higher concentrations or longer incubation times were not investigated. [2]
Pre-staining of Alhydrogel with lumogallion did not show increased cytotoxicity compared to non-stained Alhydrogel based on cell counting after co-culture with THP-1 cells. [2]

[1]. The Identification of Aluminum in Human Brain Tissue Using Lumogallion and Fluorescence Microscopy. J Alzheimers Dis. 2016 Oct 18;54(4):1333-1338.

[2]. Al adjuvants can be tracked in viable cells by lumogallion staining. J Immunol Methods. 2015 Jul;422:87-94.

[3]. Highly sensitive analytical method for aluminum movement in soybean root through lumogallion staining. J. Plant Res. 110, 305–309 (1997).

Lumogallion is a histological stain used to detect aluminum in plant and mammalian tissues. It has been used to demonstrate unequivocal presence of aluminum adjuvant in a monocytic cell line. The compound strongly binds to aluminum adjuvant particles. Pre-staining of Alhydrogel affected the z-potential of aluminum aggregates from +13±4 mV to +3±2 mV, and possibly caused a slight increase in aggregate size. After pre-staining, 4.6±0.8% of aggregates were larger than 3 μm compared to 3.8±0.2% for non-stained Alhydrogel. [2]
Lumogallion can be added directly to cells to stain intracellular aluminum adjuvants after diffusion, or aluminum adjuvants can be pre-stained prior to incubation with cells to create traceable aluminum formulations. Overnight incubation with lumogallion is suggested for staining equilibrium, although a clear signal is observed after less than one hour. [2]
Not all aluminum formulations can be stained by lumogallion; Al₂O₃ nanoparticles coated with aluminic ester showed almost no fluorescence after pre-staining, while AlO(OH) nanoparticles pellets were clearly yellow. [2]

C12H9CLN2O6S

344.722

343.987

4386-25-8

97167

Brown to reddish brown solid powder

1.75g/cm3

1.706

4.199

4

8

3

22

510

0

OTKYOGBGHUUFPC-UHFFFAOYSA-N

InChI=1S/C12H9ClN2O6S/c13-6-3-9(12(18)11(4-6)22(19,20)21)15-14-8-2-1-7(16)5-10(8)17/h1-5,16-18H,(H,19,20,21)

4-Chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzene-1-sulfonic acid

Lumogallion IREALumogallion NSC-102798 NSC102798NSC 102798

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~290.08 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)