Lysosomal exocytosis assay (Hexosaminidase)

b Hexosaminidase Assay to measure lysosomal exocytosis in macrophages

Reagents and Buffers:

·         β-N-acetylhexosaminidase substrate:

4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (Sigma, M2133)

o   Prepare 300 mM in DMSO

·         Sodium citrate- phosphate buffer pH4.5

o   17.6 mM Na₂HPO₄ + 11.2 mM NaCitrate

·         Glycine buffer pH10

o   2M Na₂CO₃ + 1.1 M Glycine

·         Cell lysis buffer:

o   HEPES 25mM, NaCl 150mM, 0.5% Triton-100, add protease inhibitor prior to use

·         RPMI 1640 medium (phenol red-free, Gibco, cat.no. 11835030)

Preparation of cells:

Seed cells at a density of 1 – 1.5 x10⁵ cells/well in a 12-well plate in 1 mL RPMI 1640 Medium supplemented with 10% FBS and 1% P/S and allow cells to attach for at least 24. Incubate at 37C, 5% CO₂.

Compound treatment:

·         Wash cells once with PBS

·         Treat cells according to your experimental plan by adding 500 µl phenol red-free medium including compound:

o   Dilute compounds in serum-free RPMI 1640 medium (phenol red-free, Gibco, cat.no. 11835030) to a final volume of 500 µL per well (12-well plate)

o   Add medium containing compounds/ controls and incubate at 37C, 5% CO2  

o   Include sham control treatment (e.g. DMSO) and a positive control treatment (e.g. Ionomycin (Sigma, I0634) at a final concentration of 5 µM for 10 min). Treatment end points should be matched

Sample Collection:

·         Following incubation, collect supernatant (contains released hexosaminidase enzyme) in 1.5 mL Eppi tubes, spin down (12.000 g, 4°C) for 10 min to remove cell debris and transfer supernatant into a new Eppi tube (supernatant sample = SN)

·         Lyse cells:

o   Wash cells with PBS

o   Add 200 µL lysis buffer, scrap cells and collect suspension in 1.5 mL Eppi tubes. Lyse by sonication (2x 20 sec, 1x 10 sec)

o   Centrifuge lysates for 10 min, 12.000 g at 4°C and transfer supernatant to fresh Eppi tubes (lysate sample = L)

·         Proceed with assay

Hexosaminidase Assay:

Perform assay in a 96 well plate, include triplicates of each sample. β-Hexosaminidase enzyme activity is measured in SN as well as L samples.

·         In a well of a 96 well plate, distribute 10 µL of cell lysate + 65 µL PBS per well and 75 µL of SN/well

·         Pipette all SN and L samples in triplicates

·         Include the following assay blank values in triplicates:

o   Medium control (Blank_SN) (75 µL phenol red-free RPMI 1640 per well)

o   lysis buffer control (Blank_L) (75 µL lysis buffer per well)

·         Dilute hexosaminidase substrate in sodium citrate buffer to a working solution of 2 mM and add 75 µL 2 mM substrate to each sample/well (final concentration 1 mM). Add 75 µL citrate buffer to the blank values/well

·         Incubate for 30 min at RT

·         Stop the reaction and enhance fluorescence with 50 µL glycine buffer per well

·         Read plate with spectrofluorimeter at 365/450 (Emission/Excitation)

Analysis:

·         Subtract the average absorbance measurement of the Blank triplicates (Blank_SN) from the individual SN sample measurements, and the average of lysate Blank measurements (Blank_L) from individual L samples.

·         Calculate the average measurement of the background-subtracted sample triplicates.

·         Calculate Factor_SN by dividing volume of deployed SN probe by total SN sample volume (V_Probe/V_Total), e.g. 75 µL/ 500 µL.

·         Calculate Factor_L by dividing volume of deployed L probe by total L sample volume (V_Probe/V_Total), e.g. 10 µL/ 200 µL.

·         Multiply each SN sample value with Factor_SN and each L sample with Factor_L.

·         Determine β-hexosaminidase enzyme release as substrate turnover in supernatants relative to lysates:

                                Substrate turnover = (ΔE_SN * Factor_SN)/(ΔE_L * Factor_L)

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