TITLE: Clusterin cellular uptake assay
AUTHORS: Patricia Yuste Checa, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to efficiently monitor Clusterin and Clusterin/Substrate uptake in different cell types, like HEK293T, iNeurons and iMicroglia.

[GUIDELINES]
To study Clusterin-A488 uptake in the presence of substrate, incubate 1 micromolar (µM) Clusterin-A488 with the corresponding amount of substrate, e.g. denatured Luciferase or Aβ aggregates, in PBS for 20 min at 37 °C or 42 °C (denatured Luciferase) in a total volume of 30 µL or 40 µL in the case of HEK293T or iNeurons, respectively. After the incubation time dilute the mix 1/10 in media and add it to the cells resulting in a final concentration of 0.1 micromolar (µM) Clusterin-A488 (5 1527).
To monitor substrate uptake e.g., denatured luciferase or Aβ aggregates, in the presence of Clusterin, mix 0.2 micromolar (µM) of denatured Luciferase-pHrodo or 0.5 micromolar (µM) of Aβ-pHrodo aggregates with the corresponding amount of unlabeled Clusterin in a total volume of 30 µL or 40 µL in the case of HEK293T or iNeurons, respectively. Dilute the mix 1/10 in media. pHrodo Red dye is pH sensitive dye which fluoresces brightly only in acidic environments and therefore can be used to specifically monitor phagocytosis and endocytosis, but substrates labeled with A488 can be also used.
The indicated Clusterin-A488 or substrate concentrations and incubation times for each cell line are tentative. These parameters should be experimentally tested to be in the linear range of the assay.

[STEPS]
SECTION: Clusterin cellular uptake assay - HEK293T cells
1. Plate 100,000 HEK293T cells per well in a 24-well plate.
SECTION: Clusterin cellular uptake assay - HEK293T cells
2. On the next day, add 5 1527 of Clusterin-A488 together with 300 µL of fresh DMEM (without fetal bovine serum, 1.5 µg in 300 µL medium) to the cells and place the cells back in the incubator.

SECTION: Clusterin cellular uptake assay - HEK293T cells
3. After 240 min incubation, place the plate on ice to stop endocytosis. 

SECTION: Clusterin cellular uptake assay - HEK293T cells
4. Wash the cells gently with cold 1x PBS.
SECTION: Clusterin cellular uptake assay - HEK293T cells
5. Add 100 µL TrypL Express Enzyme (Gibco). Incubate for few minutes on ice.

SECTION: Clusterin cellular uptake assay - HEK293T cells
6. Collect the cells with 400 µL of cold medium and transfer them to an Eppendorf tube placed on ice.

SECTION: Clusterin cellular uptake assay - HEK293T cells
7. Centrifuge at 1000 x g, 5 min, 4 °C.
SECTION: Clusterin cellular uptake assay - HEK293T cells
8. Discard the supernatant and fix the cells by resuspending the cell pellet with 200 µL 4% Paraformaldehyde (PFA) in 1x PBS. Incubate for 10 min at Room temperature.

SECTION: Clusterin cellular uptake assay - HEK293T cells
9. Centrifuge at 1000 x g, 5 min, 4 °C.
SECTION: Clusterin cellular uptake assay - HEK293T cells
10. Wash the cell pellet with 1x PBS.
SECTION: Clusterin cellular uptake assay - HEK293T cells
11. Centrifuge at 1000 x g, 5 min, 4 °C.
SECTION: Clusterin cellular uptake assay - HEK293T cells
12. Resuspend the cell pellets with 160 µL 1x PBS pH 7.2 and store at 4 °C until analyzed.

SECTION: iNeurons
13. Add 5 1527 of Clusterin-A488 to 250,000 iNeurons cultured in a well of a 12-well plate (add 2 µg Clusterin-A488 to 200 µL of fresh medium and add the mix to the well with cells containing 200 µL conditioned medium) and place the cells back in the incubator.

SECTION: iNeurons
14. After 60 min incubation place the plate on ice to stop endocytosis. 

SECTION: iNeurons
15. Wash the cells gently with cold 1x PBS.
SECTION: iNeurons
16. Add 100 µL Accutase. Incubate for 5-10 minutes on ice.

SECTION: iNeurons
17. Collect the cells with 400 µL of cold medium and transfer them to Eppendorf low binding tubes placed on ice.

SECTION: iNeurons
18. Centrifuge at 1000 x g, 4 °C for 5 min (swing-bucked centrifuge preferred).
 

SECTION: iNeurons
19. Discard the supernatant and fix the cells by resuspending the cell pellet with 200 µL 4% PFA in 1x PBS. Incubate for 10 min at Room temperature.
SECTION: iNeurons
20. Centrifuge at 1000 x g, 5 min, 4 °C  (swing-bucked centrifuge preferred).

SECTION: iNeurons
21. Wash the cell pellet with 1x PBS.
SECTION: iNeurons
22. Centrifuge at 1000 x g, 5 min, 4 °C  (swing-bucked centrifuge preferred).
SECTION: iNeurons
23. Resuspend the cell pellets with 160 µL 1x PBS pH 7.2 and store at 4 °C until analyzed.

SECTION: iMicroglia
24. Dispense 150,000 iMicroglia cells per well with 300 µL medium into a Geltrex-coated 24-well plate.

SECTION: iMicroglia
25. Add 5 5 1527 of Clusterin-A488 (1.5 µg in 300 µL medium) and place the cells back in the incubator.

SECTION: iMicroglia
26. After 30 min incubation place the plate on ice to stop endocytosis, collect the cells and transfer them to Eppendorf low binding tubes placed on ice.

SECTION: iMicroglia
27. Centrifuge at 1000 x g, 5 min, 4 °C (swing-bucked centrifuge preferred).
 

SECTION: iMicroglia
28. Wash the cell pellet with 1x PBS.
SECTION: iMicroglia
29. Discard the supernatant and fix the cells by resuspending the cell pellet with 200 µL 4% PFA in 1x PBS. Incubate for 10 min at Room temperature.
SECTION: iMicroglia
30. Centrifuge at 1000 x g, 5 min, 4 °C (swing-bucked centrifuge preferred).
SECTION: iMicroglia
31. Wash the cell pellet with 1x PBS.
SECTION: iMicroglia
32. Centrifuge at 1000 x g, 5 min, 4 °C (swing-bucked centrifuge preferred).
SECTION: iMicroglia
33. Resuspend the cell pellets with 160 µL 1x PBS pH 7.2 and store at 4 °C until analyzed.
SECTION: Uptake quantification
34. Quantify Clusterin or substrate uptake by measuring A488 or pHrodo Red intensity inside the cells by flow cytometry. If A488 is used, add 50 µL of Trypan blue solution 0.4% (refer materials section) right before measuring to quench the 488 fluorescence outside the cells.