Clusterin cellular uptake assay
Patricia Yuste Checa, F Ulrich Hartl
Abstract
This protocol details how to efficiently monitor Clusterin and Clusterin/Substrate uptake in different cell types, like HEK293T, iNeurons and iMicroglia.
Attachments
Steps
Clusterin cellular uptake assay - HEK293T cells
Plate 100,000 HEK293T cells per well in a 24-well plate.
On the next day, add 5 of Clusterin-A488 together with 300µL of fresh DMEM (without fetal bovine serum, 1.5µg in 300µL medium) to the cells and place the cells back in the incubator.
After 4h 0m 0s incubation, place the plate on ice to stop endocytosis.
Wash the cells gently with cold 1x PBS.
Add 100µL TrypL Express Enzyme (Gibco). Incubate for few minutes On ice.
Collect the cells with 400µL of cold medium and transfer them to an Eppendorf tube placed On ice.
Centrifuge at 1000x g,4°C.
Discard the supernatant and fix the cells by resuspending the cell pellet with 200µL 4% Paraformaldehyde (PFA) in 1x PBS. Incubate for 0h 10m 0s at Room temperature.
Centrifuge at 1000x g,4°C.
Wash the cell pellet with 1x PBS.
Centrifuge at 1000x g,4°C.
Resuspend the cell pellets with 160µL 1x PBS 7.2 and store at 4°C until analyzed.
iNeurons
Add 5 of Clusterin-A488 to 250,000 iNeurons cultured in a well of a 12-well plate (add 2µg Clusterin-A488 to 200µL of fresh medium and add the mix to the well with cells containing 200µL conditioned medium) and place the cells back in the incubator.
After 1h 0m 0s incubation place the plate On ice to stop endocytosis.
Wash the cells gently with cold 1x PBS.
Add 100µL Accutase. Incubate for 5-10 minutes On ice.
Collect the cells with 400µL of cold medium and transfer them to Eppendorf low binding tubes placed On ice.
Centrifuge at 1000x g,4°C,0h 0m 0s for 0h 5m 0s (swing-bucked centrifuge preferred).
Discard the supernatant and fix the cells by resuspending the cell pellet with 200µL 4% PFA in 1x PBS. Incubate for 0h 10m 0s at Room temperature.
Centrifuge at 1000x g,4°C (swing-bucked centrifuge preferred).
Wash the cell pellet with 1x PBS.
Centrifuge at 1000x g,4°C (swing-bucked centrifuge preferred).
Resuspend the cell pellets with 160µL 1x PBS 7.2 and store at 4°C until analyzed.
iMicroglia
Dispense 150,000 iMicroglia cells per well with 300µL medium into a Geltrex-coated 24-well plate.
Add 5 5 of Clusterin-A488 (1.5µg in 300µL medium) and place the cells back in the incubator.
After 0h 30m 0s incubation place the plate on ice to stop endocytosis, collect the cells and transfer them to Eppendorf low binding tubes placed On ice.
Centrifuge at 1000x g,4°C (swing-bucked centrifuge preferred).
Wash the cell pellet with 1x PBS.
Discard the supernatant and fix the cells by resuspending the cell pellet with 200µL 4% PFA in 1x PBS. Incubate for 0h 10m 0s at Room temperature.
Centrifuge at 1000x g,4°C (swing-bucked centrifuge preferred).
Wash the cell pellet with 1x PBS.
Centrifuge at 1000x g,4°C (swing-bucked centrifuge preferred).
Resuspend the cell pellets with 160µL 1x PBS 7.2 and store at 4°C until analyzed.
Uptake quantification
Quantify Clusterin or substrate uptake by measuring A488 or pHrodo Red intensity inside the cells by flow cytometry. If A488 is used, add 50µL of Trypan blue solution 0.4% (refer materials section) right before measuring to quench the 488 fluorescence outside the cells.
