Isolation of Clones with Agarose-Embedded Cloning Rings

Isolation of clones with agarose-embedded cloning rings

PROTOCOL FOR: An agarose-based cloning-ring anchoring method for isolation of viable cell clones

Saroj P. Mathupala1 and Andrew E. Sloan2

1Department of Neurological Surgery and Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA and 2Department of Neurological Surgery, Case Comprehensive Cancer Center, University Hospitals-Case Medical Center, Cleveland, OH, USA

Supplementary material for this article is available at www.BioTechniques.com/article/113079.

LEGEND ATTENTION * HINT REST

REAGENTS

 Low melting-point (LMP) agarose powder (65°C melting temperature, 25°C gelling temperature) (Cat. no. A9414; Sigma-Aldrich Corp., St. Louis, MO, USA, or equivalent)  0.05% Trypsin-EDTA (Cat. no. 25300-054; Invitrogen/Gibco, Carlsbad, CA, USA); 0.05% trypsin and 0.53 mM EDTA in Hank’s balanced salt solution; or an equivalent buffer solution free of Ca2+ and Mg2+  Dulbecco’s phosphate buffered saline (Cat. no. 14190-144, Invitrogen/Gibco, or an equivalent), free of Ca2+ and Mg2+  Complete media, supplemented with 10% serum (Cat. no. 11885- 084, Invitrogen/Gibco)

PROCEDURE

PREPARATION OF PASTEUR PIPETTES WITH BENT ENDS 1. Place the cotton-tipped pipettes in sterilization pouches. Sterilize the pipettes in dry cycle. 2. Transfer the pipettes to tissue culture hood. 3. Using a Bunsen or alcohol burner, flame the pipettes ∼1.0 cm from tip. When malleable, press the side of the tip against the “hot” upper part of the burners to make a 90° bend. The distance between tip of the pipettes and the bend should be such that it is longer than the depth of the tissue culture plates. 4. Place the prepared pipettes in an inverted position on a suitable rack (e.g., an empty Styrofoam rack used to stack 15-mL plastic tubes) in the tissue culture hood until use.

PREPARATION OF 1% (W/V) LMP AGAROSE (FILTER-STERILIZED METHOD) 5. Place 20 mL water or PBS in a 40-mL beaker equipped with a stir bar. Place on a heater-equipped stirrer. 6. While stirring, add 0.2 g LMP agarose powder. 7. Heat, monitor closely until the agarose is melted and the solution is clear (∼70°C). Do not allow the solution to boil. 8. Immediately after the solution becomes clear, transfer the beaker to tissue culture hood. Remove the plunger from a 10-mL syringe, attach a filter cartridge, and pour 10 mL of the melted agarose into the barrel. Immediately replace the plunger, and push the agarose solution into sterile 15-mL polypropylene or equivalent tubes. 9. Repeat the process to sterilize the remaining 10 mL of melted agarose. 10. Transfer the tubes to a 37°C water bath until use. *An alternative method for preparation of 1% (w/v) LMP agarose using an autoclave (this method is suitable for preparing larger volumes of sterile agarose) is as follows:  Place 50 mL water or PBS in a 100-mL wide-mouthed glass bottle suitable for preparing autoclaved media. Add 0.5 g LMP agarose powder.  Mix by swirling and autoclave in wet cycle for 15 min at 121°C.  Immediately after the autoclave cycle is completed, remove the bottle from autoclave and swirl to mix the agarose into a uniform solution.  Transfer the bottle to a 37°C water bath until use. Long-term storage of prepared sterile agarose: The prepared sterile agarose solutions can be stored at 4°C for later use. To re-melt stored agarose, place the polypropylene tubes in a boiling water bath until all agarose is melted, and then transfer to the 37°C water bath to equilibrate temperature. In case of sterilized glass bottles containing agarose, one may microwave to re-melt the agarose, or place the bottle in a shallow pan of boiling water to melt the agarose before transfer to the 37°C water bath.

PREPARATION OF 24-WELL CLONE RECEIVING PLATE 11. Add 1 mL complete media (supplemented with 10% serum) to each well of the plate, according to the number of clones that are to be lifted from the tissue culture plate. Complete media containing 10% serum is necessary, as the serum components are needed to inhibit and inactivate trypsin in the clonal cell suspensions (the preceding steps).

EMBEDDING CLONING CYLINDERS IN AGAROSE (the following describes the procedure for 100-mm diameter tissue culture plates) 12. Mark the underside of tissue culture plates to indentify cell clones suitable for lifting. 13. Transfer the plates to tissue culture hood, aspirate spent media, and wash the plates once in 10 mL room temperature PBS (free from Ca2+ and Mg2+). 14. Aspirate the PBS. Hold the plates at an angle for 10–20 s and aspirate any residual PBS. Removing residual PBS is necessary, in order to prevent dilution of agarose in the subsequent embedding step.

15. Carefully place the cloning cylinders around each clone with the curved forceps (see Supplementary Figure 1A). If necessary, reposition the cylinders around the clones. 16. Using a 1- or 2-mL pipette, add melted agarose (at 37°C) dropwise along the bottom (outer) edge of each of the cylinders (3 or 4 drops would suffice to create a seal and embed the cylinder to the plate) (see Supplementary Figure 1B). 17. Carefully replace the cover of the plates, and allow the agarose to solidify (keep the plates flat on the metal surface of the tissue culture hood for 2-5 min).

TRYPSIN-EDTA TREATMENT OF THE EMBEDDED CELL CLONES 18. With a micropipette, add 40 μL of 0.05% trypsin-EDTA into each of the cloning cylinders (see Supplementary Figure 1C). 19. Replace the lid and transfer the plate to a 37°C incubator for 2–5 min. Monitor the detachment of cells under an inverted microscope. (Once the cells take an oval shape they are ready to be detached and aspirated.) 20. Transfer the plates back to the hood. 21. Aspirate ~100 μL media from a well of the 24-well plate into a prepared Pasteur pipette (draw up until the liquid extends about 2 cm into the tip of the pipette). 22. To ease manipulation of the pipette tip inside the cloning cylinder, place the tissue culture plate at an angle in the hood (see Supplementary Figure 1D). Expirate sufficient media into the cylinder until it is filled to capacity, and slowly pipette in-and-out several times to detach the cells. 23. Transfer the entire cell suspension with the same pipette into the corresponding well in the 24-well plate (see Supplementary Figure 1E). 24. Repeat the process quickly until all clones are transferred to the 24-well plate. 25. Transfer the plate to tissue culture incubator for propagation and downstream analysis. EQUIPMENT

 Small tapered cloning cylinders (sterile, 4.7 mm interior diameter × 8 mm tall) (Cat. no. 378470100; Bel-Art Products, Pequannock, NJ, USA, or equivalent).  Non-adsorbant, cotton-tipped, 5.75-in Pasteur pipettes, (Cat. no. 13-678-8A; Fisher Scientific, Hanover Park, IL, USA), or Pasteur pipettes plugged with non-adsorbant cotton (Cat. no. 07-895; Fisher Scientific, Hanover Park, IL, USA)  Jeweler’s forceps, curved, 4.75-in (Cat. no. 08-953-F; Fisher Scientific, or equivalent)  3.5-in × 9-in sterilization pouches (Cat. no. 01-812-51; Fisher Scientific, or equivalent)  Disposable 24-well tissue culture plates, 1.0- or 2.0-mL (e.g. Cat no. 3047, Becton Dickinson, Lincoln Park, NJ, USA)  100- or 200-μL sterile micropipette (Cat. no. L-200, Rainin Instruments, LLC., Oakland, CA, USA, or equivalent)  Disposable 100- or 200-μL disposable filtered sterile tips (Cat. no. RT-L200F, Rainin Instruments, LLC., or equivalent)  15-mL sterile, disposable polypropylene or polystyrene tubes (Cat. no. 430790, Corning Inc., Corning, NY, USA, or equivalent)  Bunsen burner (Cat. no. S41898; Fisher Scientific) or alcohol burner (Cat. no. S41898, Fisher Scientific)  Heater-equipped stirrer (Model no. PC-520, Stirrer/Hotplate, Corning Inc.)  10-mL plastic or glass Luer Lock syringes (BD-disposable, Cat. no. 309604; Becton Dickinson, Lincoln Park, NJ, USA or Cat. no. 14-823-2A; Fisher Scientific)*  0.22-μm sterile Luer Lock syringe-filter cartridges (Millex, 33-mm diameter, Cat. no. SLGV033RS; Millipore Corp., Danvers, MA, USA)*

*Luer Lock syringes and syringe-filter cartridges are required to prepare sterile agarose per this protocol. However, an alternative autoclave-based method that does not require syringes or filters is also described below (see Hint, above).