How to measure degranulation assay for suspension cells?

08 March 2021 1 784 Report

Hello,

I am working with mast cells suspension cells (i.e. BMMCs and HMC), and I was wondering if anyone has any experience working with these cells. Specifically performing a degranulation assay on these cells. If anyone has a protocol they could share or any tips because these are suspension cells so after exposure to the a specific chemical, it is difficult to separate cells from supernatant.

Thank you!

Alexandr Chernov

Hello, Shereen!

Please You look at following article and protocol:

Article Measuring Mast Cell Mediator Release

Measurement of mast cell degranulation as monitored by β-hexosaminidase release

Materials

Cells

Mouse bone marrow-derived mast cells (BMMCs), primary human mast cells (HuMCs) obtained from CD34+ peripheral blood progenitor cells, or the human LAD2 mast cell line.

Media for BMMCs

RPMI1640 supplemented with 10% FBS, glutamine (4 mM), sodium pyruvate (1 mM), penicillin (100 units/ml), streptomycin (100 μg/ml), non-essential amino acids (10 ml/L) (Sigma-Aldrich), HEPES (25 mM), β-mercaptoethanol (50 μM), and mouse recombinant IL-3 (30 ng/ml) (Peprotech).

Media for HuMCs

StemPro-34 culture media (GIBCO) containing the StemPro-34 nutrient supplement as indicated by the manufacturer (GIBCO), glutamine (2 mM), penicillin (100 units/ml), streptomycin (100 μg/ml), recombinant human IL-3 (30 ng/ml) (for use in first week cultures only), IL-6 (100 ng/ml), and human stem cell factor (SCF) (100 ng/ml) (Peprotech)

Media for LAD2 cells

Mouse monoclonal anti-DNP-IgE (clone SPE-7) (Sigma-Aldrich)

Dinitrophenyl-human serum albumin conjugate (DNP-HSA) (Sigma-Aldrich)

Human myeloma IgE (Calbiochem), biotinylated using NHS-biotin (Zymed) (see Unit 5.3. support protocol 2)

Streptavidin (Sigma-Aldrich)

HEPES buffer (pH 7.4): HEPES (10 mM), NaCl (137 mM), KCl (2.7 mM), Na2HPO4·7H2O (0.4 mM), glucose (5.6 mM), CaCl2·2H2O (1.8mM), MgSO4·7H2O (1.3mM), bovine serum albumin (BSA) (0.04%)

Citrate buffer (pH 4.5): citric acid (40 mM), Na2HPO4·7H2O (20 mM)

Glycine (400 mM; pH 10.7)

Triton X-100 (0.1%)

p-nitrophenyl N-acetyl-β-D-glucosamide (PNAG) (Sigma-Aldrich)

96 well plates

15 ml polypropylene conical tubes

1.5 ml e-tube

Sonicator (e.g. Cole Parmer Ultrasonic Homogenizer)

8 or 12 tip multichannel pipettor

Table top centrifuge (e.g. Sorvall RT-6000B refrigerated centrifuge)

NOTE: All solutions are made in distilled water. All solutions and media must be filtered and keep in cold room.

Procedures

BMMCs are obtained by flushing bone marrow cells from the femurs of C57BL/6J mice (The Jackson Laboratory), then culturing the cells for 4–6 weeks in RPMI containing mouse recombinant IL-3 (30 ng/ml) (see Unit 3.23). BMMCS can also be obtained from other mouse strains by this protocol. BMMCs are generally harvested for used between 4–6 weeks of culture. Primary HuMCs are developed from CD34+-peripheral blood progenitor cells in StemPro-34 culture media containing recombinant human IL-3 (first week cultrures only), IL-6, and stem cell factor and are used between 7–10 wk after initiation of culture. LAD2 cells are cultured in StemPro-34 culture media containing human SCF (100 ng/ml). (“Generation, Isolation, and Maintenance of Human Mast Cells and Mast Cell lines” Unit) Cells are sensitized overnight with an optimal concentration (100 ng/ml) of biotinylated human IgE (for HuMCs or LAD2 cells) or mouse anti-DNP-IgE (for BMMCs). NOTE: In some case, BMMCs are cultured in SCF and IL-3 in RPMI. In this case, the results may be different from the cells cultured in IL-3 only. NOTE: The genetic background of the mice influences the extent of FcεRI-mediated mast cell mediator release. For this reason it is essential to use the same genetic background, ideally littermate controls, of knock out and wild type for direct comparisons. NOTE: All incubations are performed in a humidified 37 °C, 5% CO2 incubator unless otherwise specified. NOTE: To avoid the influence of IL-3, or where used SCF, on results, cells should be starved from 4 h to overnight, in cytokine-free medium. NOTE: Prepare adequate numbers of cells for duplicate assays and to anticipate procedural losses. NOTE: HuMCs, LAD2 and BMMCs are non-adherent cells. To replace medium or wash the cells, spin the cells at 450 x g, room temperature (RT) for 5 min and carefully remove the supernatant; treat the cells gently.

Following sensitization, wash the cells three times with 10 ml HEPES buffer (maintained at 37 °C) to remove excess IgE.

Resuspend cells in the HEPES buffer and aliquot the cells into 96 well plates (HuMCs, LAD2 cells 5,000–10,000/well, BMMCs 30,000–50,000/well). Final volume per well is 100 μl including agonist. As agonists and antagonists are added in 10μl of 10× final concentrations, cell suspensions are adjusted to accommodate this volume. For example, if the cells are treated with one agonist, the cells are adjusted such that the correct final amount of cells is added in 90 μl. If the cells are challenged with one agonist and one antagonist, then the cells are added in 80 μl. NOTE: Samples must be run at least in duplicate. To correct for background fluorescence, prepare blanks (HEPES buffer only).

Incubate cells in 37 °C for 5–10 minutes to equilibrate the cells with activation temperature.

Prepare 10× stocks of agonists or inhibitors in HEPES buffer. Then, add 10 μl of these stocks to each well. For example, to stimulate BMMCs with 100 ng/ml of DNP-HSA, prepare 1 μg/ml of DNP-HSA in HEPES buffer and add 10 μl into 90 μl of the cell suspension in the well. NOTE: For inhibitor studies, cells are preincubated with inhibitors for 10–30 min prior to the addition of the agonists. When using SCF or other agonist such as G-protein coupled receptor (GPCR) agonist, add just prior to, or concurrently with antigen.

Incubate the plate in a heating block or warm air oven at 37 °C for 30 min (without CO2).

During incubation, prepare p-nitrophenyl N-acetyl-β-D-glucosamide (PNAG) solution in citrated buffer. NOTE: Calculate the total volume required based on a volume of 10 ml per complete 96 well plate. Dissolve 35 mg of PNAG per 10 ml of citrate buffer. PNAG is poorly soluble in the citrate buffer, therefore sonication may be required to fully solubilize.

Aliquot 100 μl of PNAG solution into two new 96 well plates using a multichannel pipettor. One plate is for measurement of secreted β-hexosaminidase activity (supernatants) and the other is used to calculate the total amount (supernatants and lysates) to determine the percentage release.

After step 6, spin the plate 450 x g, at 4 °C for 5 min to stop the reaction and to ensure the cells are sedimented to the base of the wells.

Tilt the plate to an angle of 45° then CAREFULLY remove 50 μl aliquots of the cell free supernatant using a multichannel pipettor by removing from the top of where the depth of the solution is greatest. Add the aliquots into the PNAG solution and incubate the plate for 90 min at 37 °C (without CO2). NOTE: Make sure the cells are not removed then transferred into PNAG solution when you take 50 μl of supernatant as this will produce an artificially high degranulation value.

In order to calculate the total β-hexosaminidase activities, add 150 μl of 0.1% Triton X-100 solution into the 50 μl of supernatants and cells remaining in the original incubation plate. Resuspend carefully and take 50 μl of lysates and add into the PNAG solution and incubate the plate for 90 min at 37 °C (without CO2). Alternatively, add 150 μl of distilled water instead of 0.1% Triton X-100 solution and freeze the plate for over 1 hour at −80 °C. Defrost, mix by pipetting up and down then remove 50 μl to assess lysate content as above..

After incubation, add 50 μl 0.4 M Glycine buffer into each well. The appearance of yellow color indicates the extent of β-hexosaminidase activity.

Read plate absorbance at 405 nM with reference filter at 620 nm. Calculate the percentage of β-hexosaminidase activities present in the supernatants.

Calculations

Percentage degranulation = 100 × (superatant content)/(supernatant + lysate content)

However only ½ of supernatant is assayed and plate blank needs to be deducted. Therefore numerator (total supernatant reading) = 2 × (supernatnat − plate blank)

Only ¼ of lysates is used and lysates still contain ½ of supernatant. Plate blank also needs to be deducted. Therefore denominator = (total cell content prior to stimulation) = (1/2 (total supernatant − plate blank)) + (4 × (lysates − plate blank))

% release therefore = 100 × (2 × (supernatant − plate blank))/(1/2 (total supernatant − plate blank)) + (4 × (lysates − plate blank))

For example if supernatant reading = 0.4, lysates = 0.3, and plate blank = 0.04

Then % release = 100 × (2 × (0.4 − 0.04)/(0.4 − 0.04) + (4 × (0.3 − 0.04))) = 51 % release

These calculations can readily be carried out using a spread sheet such as MS-Excel.

Measurement of eicosanoid (PGD2, LTC4) generation in mast cells

Materials

Cells (BMMCs, HuMCs), media, and other materials are the same as in “Measurement of degranulation as monitored by β-hexosaminidase release from mast cells”

Prostaglandin D2-MOX EIA kit (e.g., Cayman chemical)

Leukotriene C4 EIA kit (e.g., Cayman chemical)

NOTE: PGD2 is chemically unstable so it can rapidly degrade in the presence of serum. To avoid degradation a PGD2 Methoxime enzyme immunoassay (PGD2-MOX EIA) is used which is based on the conversion of PGD2 to its stable MOX derivate. NOTE: Prepare ultra pure water for the eicosanoid measurement assay. Water must be deionized and free of trace organic contaminations. To accomplish the latteruUse an activated carbon filter cartridge or organic scavengers.

HuMCs or BMMCs are sensitized overnight with an optimal concentration (100 ng/ml) of biotinylated human IgE or mouse SPE-7, respectively, in cytokine and stem cell factor-free medium. NOTE: All incubations are performed in a humidified 37°C, 5% CO2 incubator. NOTE: Assays are performed in duplicate so prepare an appropriate number of cells to account for this and also for procedural loss.

The next day, wash the cells three times with 10 ml warm HEPES buffer to remove excess IgE.

Resuspend cells with HEPES buffer and aliquot the cells into 96 well plates or 1.5 ml e-tube (HuMCs; 200–250 cells/sample for both PGD2 and LTC4, BMMCs; 2000 cells/sample for PGD2, 100 cells/sample for LTC4). Final volume will be 100 μl. NOTE: Cell number is very critical for measurement of antigen-induced eicosanoid generation. In the case of LTC4 measurement in BMMCs, only 100 cells are needed. This low number can easily lead to inaccuracies in cell quantification. Therefore, it is recommended to use 100–1000 fold more cells and dilute the supernatants later with HEPES buffer. NOTE: To measure the generation of eicosanoids in response to other agonists such as SCF, GPCR agonist, etc, it may be necessary to first determine the optimal cell number.

Incubate cells at 37 °C for 5–10 minutes to equilibrate the cells with the activation temperature then add 1/10 of the final reaction volumes of 10× stocks of antigen in HEPES buffer. (DNP-HSA for BMMCs, Streptavidin for HuMCs (10–100 ng/ml is the optimal concentration range).

Incubate the plate at 37 °C for 20–30 min.

Spin down the plate (450 × g, 5 min, 4 °C) or 1.5 ml e-tube to recover free supernatant. Store the supernatants at −80 °C until assayed. NOTE: Eicosanoids are chemically unstable and can rapidly degrade. Samples should thus be assayed immediately after collection or stored at −80 °C until assayed.

Measure LTC4 and PGD2 in the supernatants by competitive enzyme immunoassay (Cayman Chemicals) according to the manufacturer’s instructions. The amounts of LTC4 and PGD2 are calculated based on the standard curve (10 pg/ml–1000 pg/ml). NOTE: A new standard curve must be run for each assay as results can vary depending on the development condition.

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