SCENITH assay

Protocol Name:

SCENITH Protocol (Single-Cell Energetic Metabolism Profiling by Translation Inhibition)

Target Application:

Intracellular Flow Cytometry / Metabolic Profiling

Incubation Conditions:

37°C, 5% CO2 / 4°C / Room Temperature (RT)

Step 1

Pre-heat the three metabolic drugs (C, DG, O) and puromycin at 37°C

Step 2

Label Eppendorf tubes as C, DG, O, and DG+O for each experimental condition. Aliquot 125,000 to 250,000 DCs in 100 µL into each Eppendorf tube.

Step 3

Add corresponding metabolic drugs to each tube as applicable. 5 µL / tube (C, DG, O, DG+O)

Step 4

Add 5µL Puromycin to each tube and incubate for 40 min at 37°C, 5% CO2

Step 5

Fill tubes with ice-cold FACS buffer (add 500 µL). Centrifuge at 3.3 rpm, 8 min, 4°C. Discard the supernatant and resuspend the pellet in its residual volume.

Step 6 (If surface staining is done)

Resuspend cells in 100 µL of ice-cold FACS buffer containing 10 µL/tube of surface antibody mix. Incubate in the dark for 25 min at 4°C

Step 7

Add 500 µL FACS buffer, centrifuge at 3.3 rpm, 8 min, 4°C, discard the supernatant, and resuspend the pellet in residual volume.

Step 8

Add 100 µL 1% PFA for fixation and incubate in the dark for 20 min at RT 

Step 9

Add 400 µL 1X Permeabilization Buffer and centrifuge samples at 3.5 rpm, 7 min, RT. Discard the supernatant and resuspend cells.

Step 10

Add 50 µLintracellular blocking solution with anti-puromycin-AF647 antibody (0.25 µL per tube) to all tubes.

Step 11

Incubate intracellular antibody mixture in the dark.

Step 12

Fill with 1X Permeabilization Buffer and centrifuge samples at 3.5 rpm, 7 min, 4°C. Discard the supernatant and resuspend by gentle tapping.

Step 13

Resuspend cells in Isoflow and transfer to flow cytometry tubes for analysis.

Reagent / Buffer Solution

Preparation Recipe (4 Tubes)

1% PFA

400 µL (4% PFA diluted in 1X PBS)

1X PERM Buffer

4.4 mL (10X Permeabilization Buffer from FoxP3 Kit diluted in sterile water)

BLOCKING Buffer

200 µL (1X PERM Buffer + 20% FBS)

Anti-Puromycin Mix

250 µL Blocking Solution + 1 µL Anti-Puromycin-AF647 (Provides 50 µL/tube)