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).