An Immunofluorescence Assay to Characterize and Quantify Mammalian Stress Granules

Published: January 31, 2024

Abstract

Source: Aulas, A. et al., Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules. J. Vis. Exp. (2017)

This video demonstrates an assay for characterizing and quantifying mammalian stress granules, which consist of mRNAs, RNA-binding proteins (RBPs), and various other proteins.

Protocol

1. Cell Preparation Add autoclaved coverslips to 12 wells of a 24-well plate, as indicated in Figure 1A; this can be done using a sterile Pasteur pipette attached to a vacuum to pick up each coverslip. Gently tap the coverslip on the side of the well to release the suction, allowing the coverslip to fall into the well. Plate 1 x 105 U-2 OS (U2OS) osteosarcoma cells per well at a final volume of 500 µL of medium. Move the plate side to side …

Representative Results

Figure 1: Experimental Diagrams. (A) Seed cells onto coverslips previously placed in a 24-well plate, as indicated. Treat Rows A and B for 60 min with SA or VRB using the concentration in µM as indicated. After 60 min, add CHX (final concentration: 20 µg/mL for column 2) or puro (20 µg/mL puromycin for column 4) to the drug-containing medium. Continue incubati…

Divulgazioni

The authors have nothing to disclose.

Materials

U-2 OS ATCC ATCC®HTB-96™ – commonly written as "U2OS"
– culture at 37 °C under 5% CO2 in 10% FBS/DMEM
Dulbecco's Modification of Eagle's Medium Corning 10-013-CV – abbreviated DMEM
– contains 4.5 g/L glucose, pyruvate, and phenol red
– supplemented with 10% FBS, 10 mM HEPES, and penicillin/steptomycin
– prewarm prior drug(s) dilution
– prewarm prior drug(s) dilution
Fetal Bovine Serum Sigma F2442-500ml – abbreviated FBS
– used to supplement media
HEPES (1 M) Thermo Fisher Scientific 15630-080 – used to supplement media
Penicilline Streptomycine Sigma P0781-100ml – used to supplement media
24 well plate Costar 3524
Lab tissues KIMTECH SCIENCE 34120 – commonly called "kimwipes"
Coverglass for Growth Cover Glasses Fisher Scientific 12-545-82 – autoclaved before used
– keep sterile in the tissue culture hood
Sodium (meta)arsenite ≥90% Sigma S7400-100G – commonly called sodium arsenite and abbreviated SA
– dissolution in water at 1 M concentration, then dilute to 100 µM as a working stock
Vinorelbine TSZ CHEM RS055 – abbreviated VRB
– dissolve in water to 10 mM
Puromycin Sigma P9620 – used to assess translation level (ribopuromycylation)
– used to assess SG connection with active translation
– dilute in water to 10 mg/mL
Emetine dihydrochloride hydrate Sigma E2375 – used in combination with puro to assess general translation level
– used to assess SG connection with active translation
– dilute in water to 10 mg/mL
Cycloheximide Sigma C4859 – abbreviated CHX
– used to assess SG connection with active translation
– dilute in water to 10 mg/mL
Phosphate Buffered Saline Lonza / VWR 95042-486 – abbreviated PBS
– wash buffer for immunofluorescence
Paraformaldehyde reagent grade, crystalline Sigma P6148-500G – make a 4% solution in hot PBS in fume hood, stir until dissolved
– aliquots can be stored at – 20 °C for several months
– hazardous, use ventilation
– discard in special waste
Methanol, ACS BDH / VWR BDH1135-4LP – pre-chilled to -20°C before use
– discard in special waste
Normal Horse Serum Thermo Fisher Scientific 31874 – abbreviated NHS
– dilute to 5% in PBS
– add sodium azide for storage at 4°C
– blocking solution for immunofluorescence
Ethanol, Pure, 200 Proof (100%),USP, KOPTEC Decon Labs / VWR 89125-188 – dilute to 70% with water
Fisherbrand Superfrost Plus Microscope Slides Fisherbrand 12-550-15
Mouse anti G3BP1 antibody (TT-Y) Santa Cruz sc-81940 – store at 4 °C
– use at 1/100 dilution
Rabbit anti eIF4G antibody (H-300) Santa Cruz sc-11373 – store at 4 °C
– 1/250 dilution
Goat anti eIF3η (N-20) antibody Santa Cruz sc-16377 – eIF3η is also known as eIF3b
– store at 4 °C
– 1/250 dilution
Cy2 AffiniPure Donkey Anti-Mouse IgG (H+L) Jackson Immunoresearch 715-225-150 – reconstitute in water as per manufacturer's instructions then store at 4°C
– 1/250 dilution
Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L) Jackson Immunoresearch 711-165-152 – reconstitute in water as per manufacturer's instructions then store at 4°C
– 1/2500 dilution
Cy5 AffiniPure Donkey Anti-Goat IgG (H+L) Jackson Immunoresearch 705-175-147 – reconstitute in water as per manufacturer's instructions then store at 4°C
– 1/250 dilution
Hoechst 33258 solution Sigma 94403-1ML – incubate with secondary antibodies-stock solution 0.5 mg/mL in dH20, protect from light- Store at 4 °C
20 x SSC buffer Thermo fisher Ambion AM9763 – dilute to 2X SSC using RNase Free water (DEPC treated)- store at room temperature- wash buffer for FISH
Slide mounting media home made / – mix 5 g of "cold-soluble" poly(vinyl alcohol) in 20 ml of PBS- mix by sonication, followed by stirring overnight at RT- Add 5 ml of glycerol and 0.2 ml of 20 % sodium azide, and stir for 16 h at RT- centrifugation at 20,000 g for 20 min, discard large pellet- aliquot viscous liquid, long-term storage at -20°C, 1 week at 4°C
Parafilm "M" Sigma P7793-1EA – usually referred as "parafilm"
Poly(vinyl alcohol) Sigma P-8136-250G – reagent to make vinol
Glycerol Sigma G5516-100ML – reagent to make vinol
Sodium azide Fisher Scientific S2002-5G – preservative agent for blocking solution and vinol
– make a 20 % dilution in water

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Citazione di questo articolo
An Immunofluorescence Assay to Characterize and Quantify Mammalian Stress Granules. J. Vis. Exp. (Pending Publication), e21894, doi: (2024).

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