Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

NOTE: Male C57BL/6 mice (5–7 weeks old) are from Charles River Laboratories International, Inc. (St-Constant, Quebec, Canada.) and manipulated according to protocols approved by the CHU Sainte-Justine Animal Care Committee. Mouse bronchial tissue explants provide a good source of primary bronchial epithelial cells for investigating the proapoptotic effects of LMPs on epithelial cells. This protocol describes the in vitro generation of LMPs, as well as a method for detecting apoptotic epithelial cells on LMPs-treated bronchial tissue explants. This protocol consists of 3 sections.

1. LMPs Production and Characterization

NOTE: To prevent contamination, ensure that all materials used in this experiment are sterile or autoclaved. Perform all steps at RT in a biological safety cabinet under sterile condition, unless otherwise indicated.

1.1) Stimulation and Collection of MPs⁹

1. Thaw an aliquot of 10 million CEM T cells in a 37 °C water bath. Dilute in 10 ml pre-warmed serum-free hematopoietic medium such as X-VIVO, in a 15 ml sterile tube and centrifuge at 200 g x 5 min. Aspirate the supernatant and resuspend cells in 5 ml pre-warmed medium.
2. Transfer cells into a T75 tissue culture flask (for suspension cells) with 15 ml pre-warmed hematopoietic medium such as X-VIVO and incubate for 4 days in a humidified incubator at 37 °C with 5% CO₂ .
3. After 4 days, transfer all the culture medium and cells into a T175 tissue culture flask containing 100 ml fresh medium. Continue incubating the cells for about 72 hr under the same conditions until they have grown to a density of 2 million cells/ml.
4. Evenly split cells between four T175 flasks each containing 150 ml fresh medium and continue cell culture until cells have grown (approximately 48 hr incubation) to a density of 2 million/ml.
5. Collect cells from each flask by centrifugation at 200 x g for 5 min and resuspend 300 x 10⁶ cells into a new T175 flask containing 150 ml fresh medium, to maintain the 2 million/ml cell density .
6. Add actinomycin D (dissolved in DMSO at 2 mg/ml) to the medium at a final concentration of 0.5 µg/ml and incubate for 24 hr.
7. Transfer all the culture medium into 50 ml conical tubes and spin down the cells at 750 x g for 5 min. Transfer the supernatant into 50 ml conical tubes and centrifuge at 1,500 x g for 15 min to remove large cell fragments.
8. Transfer the supernatant into a 250 ml bottle and ultracentrifuge at 12,000 x g for 50 min. Discard the supernatant and collect pellets.
9. Wash LMPs-enriched pellets with 40 ml sterile PBS in a 50 ml tube by centrifugation at 12,000 x g for 50 min. Repeat this step twice.
10. Collect the last wash supernatant; it will be used as vehicle control. Suspend the LMPs pellets in 1 ml of PBS and transfer into a 1.5 ml sterile microtube. Aliquot and store isolated LMPs at -80 °C (to avoid multiple free-thaw cycles).

1.2) Characterization of MPs via FACS Analysis ⁴

1. Prepare 2 samples of annexin buffer, 1 with and another without CaCl₂: Hepes 10 mM, NaCl 140 mM, plus or minus 5 mM CaCl₂.
2. Filter annexin buffer and FACS flow sheath fluid using a 0.22 µm filter to remove particles.
3. Dilute 1 µl of LMPs in 44 µl of annexin buffer with 5 mM CaCl₂ into a FACS tube. Prepare another tube with 1 µl of LMPs in 44 µl of annexin buffer without CaCl₂ (negative control).
4. Add 5 µl of annexinV-Cy5 in each tube and mix well. Incubate for 15 min at RT in the dark. Stop the reaction by diluting the mix with 400 µl of FACS flow sheath fluid in each tube.
5. Add 10 µl (200,000 beads) of 7 µm counting beads suspension as an internal standard in each tube to obtain an absolute count.
6. Establish gates of relative size (FSC-H, PMT E00, log scale) and relative granularity (SSC-H, PMT 325, log scale) dot plot on the flow cytometer using size-calibrated fluorescent beads of 1 µm (gate 1) and counting beads gate at 7 µm (gate 2).
7. Analyze the LMPs sample on FSC-H /SSC-H plot using the established gates and FL-4 channel for annexin (PMT 765, log scale) dot plot, by acquiring a signal until 20,000 counting beads are reached in gate 2.
8. Determine the positive annexinV events of LMPs in annexin buffer containing CaCl₂, and then subtract the events of LMPs in annexin buffer without CaCl₂ (negative control).
9. Calculate the absolute number of MPs based on the following equation:
   

1.3) Determination of MP Protein Concentration (Bradford Assay)

1. Prepare 5 serial dilutions of a protein standard from 1.25 to 20 µg/ml. Pipette 800 µl of each standard and sample solution into a clean test tube in duplicate. Add 200 µl of Bradford dye reagent to each tube. Mix well, then incubate at RT for 5 min.
2. Measure absorbance at 595 nm. Determine the protein concentration of LMPs using the linear regression of standard curve.

2. Bronchial Tissue Explants and LMPs Treatment

NOTE: Pay special attention to the sterile working environment, and aseptically prepare the solutions and medium used in following experiments. To prepare the Complete Healing Medium, add 1 ml of Tissue Healing Medium Supplements with Serum (thawed on ice) to 100 ml Tissue Healing Medium and mix well.

2.1) Preparation of Bronchial Tissue Explants

1. Before culturing, scratch 6 areas of 1 cm² each at the edge of the surface of each 100 mm tissue culture dish with a scalpel blade. Coat each scratched 100 mm tissue culture dish with 2 ml of the culture dish coating solution, and incubate the dish in a humidified CO₂ incubator O/N at 37 °C. Vacuum aspirate the surplus solution and fill the dish with 15 ml of Tissue Washing Medium.
2. Euthanize C57BL/6 mice (5 to 7 weeks old) by CO₂ inhalation according to protocols approved by the animal care ethics committee.
3. Aseptically dissect lung tissue with scalpel, Dumont super fine tweezer, and surgical scissors. Carefully remove parenchyma and blood vessels. Place lung tissue into ice-cold Tissue Washing Medium for transport to the laboratory, if applicable.
4. Further dissect bronchus submerged in the Tissue Washing Medium and separate the bronchus with a diameter of 1 to 2.5 mm from peripheral lung tissues. Slice bronchial tissues into ~5 mm thick bronchial rings with a scalpel.
5. Use a scooping motion with the sterile curved microdissecting forceps to pick up the bronchial fragments and place them onto the scratched areas of the dishes.
6. Remove the Tissue Washing Medium, and incubate the fragments at RT for ~5 min to allow them to adhere to the dishes.
7. Add 10 ml of Complete Healing Medium to each dish and place them in a controlled atmosphere modular incubator chamber. Flush the chamber with high O₂ gas mixture (70% O₂, 25% N₂ and 5% CO₂,). Place the chamber in a benchtop orbital incubator and shake it at 37 °C. Shake the chamber for 24 hr at 10 cycles per minute to allow the medium to flow intermittently over the fragments.
8. After 24 hr incubation, observe the tissue explants under a phase-contrast inverted light microscope. Select bronchial explants with complete, fine hair movement and lively bronchial epithelium for subsequent LMPs treatment.

2.2) LMPs Treatment

1. Prepare complete growth medium as follows: thaw growth medium supplements with serum and fibroblast inhibitor on ice. Add 1 ml of the growth medium supplements with serum and 200 μl fibroblast inhibitor to 100 ml of growth medium; mix thoroughly. Warm the complete growth medium at 37 °C for 10 min prior to use.
2. Dilute isolated LMPs in a new sterile eppendorf tube with PBS to prepare a LMPs stock at a concentration of 800 µg/ml.
3. Add 0.5 ml of Complete Growth Medium to each well of a 12-well tissue culture plate.
4. Transfer the selected bronchial explants with the curved microdissecting forceps from the previous protocol (section 2.1) to each well of the tissue culture plate.
5. Label the culture plate appropriately to identify LMPs treatment wells and control wells. Add 25 µl LMPs stock into each LMPs treatment well (for a final concentration of 40 μg/ml) and 25 µl control vehicle (see LMPs production) to the control wells.
6. Continue the incubation in a controlled atmosphere modular incubator chamber at 37 °C with gentle shaking.
7. After 24 hr, wash explants 3 times with PBS and proceed to the next step (4% paraformaldehyde [PFA] fixation).

3. Histopathological Examination

3.1) Prepare the Following Solutions Before Proceeding to the Next Steps

1. Prepare 1x PBS buffer by mixing 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄, pH 7.4.
2. To prepare 4% PFA, dissolve 20 g of PFA in 400 ml of water, heated at 60 °C with stirring; add a few drops of 10 M NaOH to clear the solution. Next add 1x PBS buffer and adjust the volume to 500 ml and pH to 7.4. Filter and aliquot; store at -20 °C.
3. Prepare the following dehydration or rehydration reagents; 100%, 90%, 70%, 50% ethanol and xylene.

3.2) Explant Fixation and Tissue Section Deparaffinization

1. Place each explant in a labelled microcentrifuge tube with 1.5 ml of 4% PFA and incubate O/N at 4 °C. Rinse the explants twice with 1x PBS.
2. Dehydrate explants through an alcohol series (70% ethanol: 3 times 30 min each; 90% ethanol: 2 times 30 min each; 100% ethanol: 3 times 30 min each; then xylene: 3 times 20 min each). Perform all steps at RT in a fume hood.
3. Imbed tissue explants in paraffin at 58 °C in an oven. Prepare 5 µm thick tissue sections using a rotary microtome.
4. Float the sections in a 56 °C water bath, and then mount the sections onto labeled histological slides. Place the slides in manual staining racks and dry at 65 °C for 1 hr. Allow the slides to cool at RT.
5. Dip the racks in 4 consecutive stain dishes containing xylene for 10 min each to remove paraffin. Dip the racks in an ethanol series to remove xylene: 100%, then 95%, then 80%, then 70%, then 50% ethanol (5 min for every step). Rinse the racks with tap water for 5 min to remove ethanol.

3.3) Hematoxylin and Eosin (H&E) Staining 

1. Continue working with the fixed tissue sections; place the rack into a staining dish filled with Mayer's Hematoxylin for 15 min. Rinse the rack with tap water to remove Hematoxylin for 20 min.
2. Place in distilled water for 30 sec.Place in 95% ethanol for 30 sec. Place in Eosin Y solution staining dish for 1 min. Dehydrate through 2 changes of 95% ethanol, 100% ethanol, and xylene for 2 min each.
3. Perform a quick check under a microscope to ensure that excess eosin is removed. Place 2 to 3 drops of Mounting Medium (Fisher SP15-100) onto each slide, then cover with a cover glass.

3.4) In Situ Cell Death Detection: TUNEL Assay

1. Before beginning, prepare proteinase K working solution: 20 µg/ml in 10 mM Tris/HCl, pH 7.4.
2. Repeat steps 1 to 5 of section 3.2 (Explant fixation and tissue section deparaffinization). Rinse the slides with deionized H₂O.
3. Immerse the slides with 1x PBS for 10 min. Drain the excess PBS. Incubate tissue sections for 30 min at RT with proteinase K working solution. Rinse slides twice with 1x PBS.
4. Perform the TUNEL assay as described in the Instruction Manual of cell death detection kit. Mount using mounting medium, and coverslip manually with glass coverslips.
5. Analyze samples under a light microscope. Use Image Pro 4.5 to analyze the apoptotic cells in brown color.