Labile Zinc Measurement by Flow Cytometry

A. Materials required:

B. Preparation of Stock for Fluozin-3-AM and Zinpyr-1:

Fluozin-3-AM: 

Stock preparation: The lyophilized vial contains 100 µg of Fluozin-3 AM. To prepare a stock of 1 mM, add 102 µl of sterile DMSO to the vial. Mix it well with the pipette. Spin down and make 10 µl aliquots. Keep in -20°C, desiccate and protect from light. 

Zinpyr-1 (ZP-1): 

Stock preparation: The lyophilized vial contains 5 mg of ZP-1. Add 2.427 ml of sterile DMSO to prepare 2.5 mM stock. Mix it well with the pipette. Spin down and make 10 µl aliquots. Keep in -20°C, desiccate and protect from light. 

C. Protocol:

    (i) Seeding of Caco-2 cells:

1. Culture Caco-2 cells in DMEM (Hyclone) supplemented with 10% FBS, 1X PSG and 1X non-essential amino acids.
2. Seed Caco-2 cells at 50,000 cells per well in 48 well-plate. 
3. These cells adhere well after two days of seeding. Treatment has to be done when cells are ~70-80% confluent.

     (ii) Cells processing for flow cytometry:

1. At the time of processing, remove media and wash the cells in plate once with 300 µl 1X PBS. 
2. Add 80 µl trypsin per well to detach cells and incubate at 37°C for 3 minutes followed by addition of 80 µl of trypsin inhibitor.
3. Resuspend cells in DMEM without phenol red supplemented with 2 mM L-glutamine (staining medium).
4. Centrifuge the cells at 800 x g for 5 minutes at room temperature (RT).
5. Discard the supernatant and dislodge the pellet by gentle vortexing for 10 seconds. 
6. Prepare 5 µM Fluozin-3 AM or 2.5 µM Zinpyr-1 (ZP-1) in the staining media at 100 µl per tube. 
   (Note: For ZP-1, staining medium containing 1 mM EDTA was added to chelate any extracellular zinc during staining. Do not use EDTA for Fluozin-3 AM.) 
7. Incubate for 30 minutes at 37°C in CO₂ incubator and mix every 10 min. After 30 minutes, add 10 µl of fixable viability stain eFluor780 (pre-diluted 1:500 in staining medium) per sample and incubate for further 10 minutes at 37°C in the CO₂ incubator. 
8. Wash cells once by adding 500 µl FACS buffer (PBS containing 0.25% FBS) to each tube and centrifuge the cells at 800 x g for 5 minutes at RT.
9. Discard the supernatant and dislodged the pellet by gentle vortexing for 10 seconds. Add 1.5 ml of   FACS buffer per tube.
10. Acquire the cells in flow cytometry.

    (iii) Cells acquisition:

1. To adjust flow cytometry settings for SSC-A, FSC-A and FITC, keep a control tube and set the parameters.
2. Keep one unstained tube to determine the location of FITC-negative population (to avoid any background fluorescence).
3. Gate cells based on their scatter properties in a SSC-A vs FSC-A plot.
4. After this, we need to gate the live cell population in a FSC-A vs APC-Cy7-A plot.
5. Gate FITC-positive population in a FSC-A vs FITC-A plot within the live cell population.
6. Acquire all samples keeping the same settings throughout the experiments.
7. Analyze data using FlowJo software. 
8. Load the data into the workspace of the software. 
9. Click on one of the sample and draw a polygon gate encompassing the population of interest in the similar manner we used for acquiring. Briefly, gate the population based on SSC vs FSC, then gate live cells followed by FITC-positive population).
10. Copy the settings to all samples and add statistics which includes fluorescence intensity, CV or frequency within a parent population, mean, median and geometric mean.

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