A Cell-Based Assay to Assess the Tau Protein Uptake by Microglia

Overview

This video showcases a cell-based assay quantifying Tau uptake by murine microglia using pH-sensitive fluorescent-labeled Tau aggregates, analyzed via fluorescence-activated cell sorting for a quantitative assessment.

Protocol

1. BV-2 Microglia Cells Culture

NOTE: Handle BV-2 cells under Biosafety Level 2 containment. The BV-2 cell line produces an enveloped recombinant ecotropic retrovirus (capable of infecting murine cells only); such viruses are known for their in vitro transforming ability and in vivo tumorigenic potential.

1. Culture BV-2 cells in high glucose Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM L-Glutamine (referred to as culturing medium from now on) by seeding cells at 4 x 10⁴ cells/mL.
2. Maintain cultures in a humidified atmosphere of 5% CO₂ at 37 °C.
   NOTE: the cells grow loosely attached and in suspension.

2. Label Recombinant Tau Aggregates with pH-sensitive Fluorescent Dye

NOTE: Tau aggregates were prepared as described in Apetri et al. with the difference that no Thioflavin T (ThT) was added to the reaction buffer. Aggregated samples were collected in 1.5 mL centrifuge tubes. The final fluorescence signal was checked by mixing 118 µL of the pool sample with 12 µL of a 50 µM ThT solution. Aggregates were separated by centrifuging the aggregation reaction mixture at 20,000 x g for 1 h at 4 °C. The supernatant was analyzed by SEC-MALS to confirm that all the monomeric tau was converted into aggregates. Pellets (tau aggregates) were snap-frozen and stored in a freezer at -80 °C.

1. Resuspend tau aggregates in 0.1 M sodium bicarbonate buffer (NaHCO₃) at pH 8.5 to a concentration of 1 mg/mL (~ 20 µM).        
   NOTE: The concentration of tau aggregates is based on the initial monomers concentration as assessed by the absorption of tau monomers at 280 nm using an extinction coefficient of 0.31 mLmg^-1cm^-1.
2. Sonicate the resuspended aggregates using a probe sonicator while keeping them on ice to avoid overheating.
   1. Use an amplitude of 65% (with a sonicator of power 250 W).
   2. Perform 8 pulses of 3 s with pauses of 15 s between pulses to avoid overheating.
3. Prepare an 8.9 mM stock solution of pH-sensitive dye (henceforth referred to as pH dye) in dimethyl sulfoxide (DMSO) following manufacturer's instruction.
   NOTE: Always prepare a fresh solution and use it only on the day it is prepared.
4. Add 10 moles of dye per mole of protein to a final dye concentration of 0.2 mM.
5. Mix by gently pipetting up and down.
6. Incubate the reaction mixture for 45–60 min at room temperature, protected from light.
7. In the meantime, assemble a cross-linked dextran gel desalting column following manufacturer's instructions.
8. Equilibrate the column with 25 mL of 0.1 M NaHCO₃ buffer pH 8.5 containing 3% DMSO. Discard the flow through.
9. Add the product of the tau aggregate labeling reaction to the column in a total volume of 2.5 mL. If the sample is less than 2.5 mL, add buffer until a total volume of 2.5 mL is achieved.
10. Let the sample enter the packed gel completely, discard the flow-through.
11. Elute with 3.5 mL of 0.1 M NaHCO₃ buffer pH 8.5 containing 3% DMSO and collect the eluate in 4 equivalent fractions in 2 mL tubes.
12. Determine protein concentrations of the 4 fractions by bicinchoninic acid (BCA) assay.
13. Store the labeled protein in a -20 °C freezer.

3. Uptake Assay with Fluorescence-activated Cell Sorting (FACS) Read-out

1. Day 1 – Seed the Cells
   1. Wash BV-2 cells in the flask by removing culturing medium and adding 1x phosphate-buffered saline (PBS).    
      NOTE: Washing volume will vary based on the size of the cell flask used. For example, for a T175 flask, wash with 10 mL of 1x PBS.
   2. Remove PBS from the flask and detach cells by incubating with trypsin-ethylenediaminetetraacetic acid (EDTA) 0.05% at 37 °C and 5% CO₂ until the cells detach from the flask (approximately 5 min).  
      NOTE: Volume of trypsin-EDTA 0.05% depends on the size of the cell flask used. For example, for a T175 flask, use 2 mL of trypsin-EDTA 0.05%.
   3. Resuspend cells in culturing medium by pipetting up and down three to five times.
      NOTE: Volume of culturing medium varies based on the size of the cell flask used and thus total number of cells in the flask. For example, for a T175 flask, use 8 mL of culturing medium.
   4. Count cells and create a cell suspension with a final concentration of 1 x 10⁵ cells/mL in culture medium containing 200 μg/mL heparin.
   5. Plate 250 µL of cell suspension (2.5 x 10⁴ cells) per well in a 96-well tissue culture flat bottom plate.
   6. Incubate plate overnight at 37 °C and 5% CO₂.
2. Day 1 – Prepare Immunocomplexes
   1. Thaw pH dye-tau on ice.
   2. Prepare 65 μl per condition of a 500 nM solution of pH dye-tau aggregates in serum-free medium (SFM) (high glucose DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 200 µg/mL of heparin).
   3. Prepare antibody dilutions in 65 µl of SFM at a concentration double the final. Mix pH dye-tau aggregates and antibodies in a 96-well u-bottom plate. The final volume per condition is now 130 µl, and the pH dye-tau aggregates concentration is 250 nM. Seal the dilution plate and incubate overnight at 37 °C.
3. Day 2 – Immunocomplexes Uptake
   1. Remove the culturing medium from BV-2 cells. Wash cells once with 100 µL room temperature 1x PBS.
   2. Transfer 125 µL of immunocomplexes to the cells using a multichannel pipette. Incubate the cells with the immunocomplexes for 2 h at 37 °C and 5% CO₂.
   3. Remove the incubation medium from the cells and discard it. Wash cells once with 100 µL room temperature 1x PBS.
   4. Remove 1x PBS and treat cells with 50 µL trypsin-EDTA 0.25% for 20 min at 37 °C and 5% CO_2.
   5. Add 200 µL of culturing medium and resuspend well by pipetting up and down to detach the cells. Transfer cells to a 96-well U-bottom plate. Centrifuge plate at 400 x g for 5 min at 4 °C.
   6. Put the plate on ice, remove culturing medium and wash cells twice by resuspending the cell pellets in 150 µL ice cold 1x PBS. Centrifuge plate at 400 x g for 5 min at 4 °C.
   7. Put cells on ice, remove added 1x PBS and wash them by resuspending cells pellets in 150 µL cold FACS buffer (1x PBS, 0.5% bovine serum albumin (BSA), 2 mM EDTA). Centrifuge plate at 400 x g for 5 min at 4 °C.
   8. Put cells on ice, remove added FACS buffer and resuspend cells in 200 µL cold FACS buffer.
   9. Analyze samples immediately by FACS acquiring 2 x 10⁴ events in the live cells gate (see step 4.1).

Materials

Name	Company	Catalog Number	Comments
BV-2 cells	ICLC Interlab Cell Line Collection	ATL03001
Phosphate Buffered Saline (PBS) (1X)	Gibco	10010-015
Trypsin-EDTA 0.05%	Gibco	25300-054
DMEM 4.5 g/dl glucose	Gibco	41966-029
Fetal Bovine Serum	Gibco	10091-148
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122
L-Glutamine 200mM	Lonza	17-605E
EasYFlask	Nunc	156499 / 159910
pHrodo Green STP ester	Life Technologies	P35369
Sodium Bicarbonate pH 8.5 100 mM
DMSO	Sigma	D2650-100ml
PD10 columns	GE Healthcare	17-0851-01
BCA Protein Assay Kit	Thermo Fisher Scientific	23225
Greiner CELLSTAR multiwell culture plates	Greiner	665180
Falcon 96-Well Assay Plates	Falcon	353910
Heparin	Sigma	H3393-50KU
Trypsin-EDTA 0.25%	Sigma	T4049-100ml
BSA	Sigma	A7030-100G
EDTA 0.5M, pH8