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Protocol

Culturing and Transfecting Zebrafish PAC2 Fibroblast Cells Niklas Senghaas1 and Reinhard W. Köster Helmholtz Zentrum München, Institute of Developmental Genetics, D-85764 Munich-Neuherberg, Germany

INTRODUCTION The use of zebrafish (Danio rerio) as a model organism has seen rapid growth in recent years. Numerous new techniques have emerged for zebrafish, and many protocols used in other model organisms have been adapted to fit the special needs of this species. Zebrafish cell lines, however, have not been studied to a great extent, and their applications remain limited. The PAC2 fibroblast line, isolated from 24-h post-fertilization zebrafish embryos, is one of the few available lines. Here, we provide a basic set of methods for maintenance and handling of PAC2 cells, as well as general procedures for transfection, immunocytochemistry, and protein extraction.

RELATED INFORMATION Further information on the PAC2 fibroblast line can be found in Culp (1994), Amsterdam et al. (1999), and Chen et al. (2002).

MATERIALS CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with , and

recipes for reagents marked with .

Reagents Antibodies, primary and secondary (for immunocytochemistry only) DMSO (dimethyl sulfoxide; ≥99.9%; Sigma D8418) DNA plasmid of interest (for transfection only) Dulbecco’s modified Eagle’s medium, liquid, high glucose (D-MEM; 1X; GIBCO/Invitrogen) Opti-MEM I reduced serum medium, with L-glutamine, liquid (1X; GIBCO/Invitrogen) may be used as an alternative.

Fetal bovine serum, EU-approved (FBS; e.g., GIBCO/Invitrogen or PAA A15-101) (optional; see Step 3) FuGENE HD (Roche Applied Science) (for FuGENE HD transfection only) IPN150 (for protein extraction only) Mounting medium (e.g., Aqua-Poly/Mount; Polysciences, Inc. 18606) (for immunocytochemistry only) Nanofectin kit (PAA) (for Nanofectin transfection only) Normal goat serum (NGS; e.g., GIBCO/Invitrogen, PAA) (for immunocytochemistry only) PAC2 medium Paraformaldehyde (4%, prepared in PTW buffer) (for immunocytochemistry only) Phosphate-buffered saline (PBS; pH 7.3) TEN (for protein extraction only)

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Corresponding author ([email protected]) Cite as: Cold Spring Harb Protoc; 2009; doi:10.1101/pdb.prot5235

© 2009 Cold Spring Harbor Laboratory Press

www.cshprotocols.org

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Vol. 4, Issue 6, June 2009

Trypsin-EDTA (GIBCO/Invitrogen) Tween 20 (Sigma-Aldrich P7949) (for immunocytochemistry only) Zebrafish PAC2 fibroblast cells

Equipment Cell scraper (e.g., Sarstedt) (for protein extraction only) Centrifuge Coverslips (for immunocytochemistry only) Cryotubes, 2-mL (Nunc) Dishes, four- or 24-well Freezing container (Nalgene 5100-00001) Ice Imaging chambers Customize as described in In Vivo Time-Lapse Imaging of Zebrafish Embryonic Development (Distel and Köster 2007) or obtain commercially (e.g., MatTek; http://www.glass-bottom-dishes.com/).

Incubator preset to 28°C Liquid nitrogen Microcentrifuge preset to 4°C Microscope, inverted Petri dishes, 35-mm and 10-cm Platform shaker Tissue culture hood, sterile Tubes, microcentrifuge, 1.5-mL Tubes, plastic, 15-mL Vortexer

METHOD Maintenance and Passaging of PAC2 Cells Perform all procedures under sterile conditions. 1. Incubate zebrafish PAC2 fibroblast cells (Fig. 1) in PAC2 medium in room atmosphere at 28°C. A CO2-rich atmosphere is not required.

FIGURE 1. Brightfield image of a single zebrafish PAC2 fibroblast cell.


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Cold Spring Harbor Protocols

2. Passage the cells when they reach 100% confluency (~1.4 × 107 cells/10-cm Petri dish): i.

Remove the culture medium. Add 10 mL of 1X PBS. Shake the dish gently.

ii. Remove the PBS. Add 1-2 mL of trypsin-EDTA to the cells. Incubate for ~10-15 min at 28°C

until all cells are detached from the plastic surface of the dish. Extensive trypsinization can harm the cells; monitor the plates under a microscope to avoid overdigestion. Accelerate detachment by carefully tapping the dish against a solid surface (e.g., a microscope stage). See Troubleshooting. iii. As soon as all cells are detached, add PAC2 medium to a total volume of 10 mL. Transfer

the suspension to a 15-mL tube. Adding medium inactivates the trypsin. iv. Centrifuge the cells at 1000-2000g for 1-2 min. Remove the supernatant carefully. The pellet is relatively small and can easily be overlooked. v. Resuspend the cells in a volume according to the desired dilution. Do not dilute more than 1:5. vi. Transfer the diluted cells to a new sterile culture dish. Culture as described in Step 1.

Cryopreservation and Thawing of PAC2 Cells 3. In a separate tube, prepare PAC2 medium (or FBS) containing 10% DMSO. Allow to cool to room

temperature. Mixing the DMSO with medium or FBS produces an exothermic reaction that can harm the cells. 4. Detach cultured cells from the surface of the dish as described in Steps 2.i-2.iv. 5. Resuspend the cells in 4 mL of PAC2 medium or FBS supplemented with 10% DMSO (from

Step 3). Transfer the suspension to two 2-mL cryotubes. 6. Cool the cryotubes at a constant rate of -1°C/min in a Nalgene freezing container according to the

manufacturer’s instructions. 7. Store the cells in liquid nitrogen. 8. To start new cultures from cryopreserved stocks, thaw the cells at room temperature. 9. Transfer the suspension from the cryotube to a 15-mL tube containing 8 mL of PAC2 medium. 10. Pellet the cells by centrifugation at 1000-3000g for 1-2 min. Remove the supernatant. 11. Resuspend the pellet in 4 mL of PAC2 medium. Transfer the suspension to a 35-mm Petri dish. It is important to use the smaller 35-mm Petri dish to achieve a reasonable growth rate. 12. (Optional) After 24 h, wash the cells with 10 mL of PBS. This step is only necessary if many dead and detached cells are observed. 13. As soon as the cells are 100% confluent, passage as described in Step 2.

Transfection PAC2 cells showed no expression after transfection with calcium phosphate, whereas Effectene (QIAGEN) transfected the cells at a very low efficiency. Protocols are presented here for transfection with FuGENE HD and Nanofectin, both of which produced transfection rates in the 5%-10% range. FuGENE is easier to handle and is used more commonly in laboratories, whereas Nanofectin is less expensive. For each reagent, the transfection is performed as described in the manufacturer’s manual. 14. Passage the cells as described in Steps 2.i-2.iv. 15. Dilute the cells 1:3. Plate them in a dish suitable for the desired application (Table 1). Allow the

cells to attach overnight.


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Table 1. Transfection conditions for zebrafish PAC2 fibroblast cells Dish type

Cell number Cell suspension Total vol. DNA (µg) (×106) (mL) (mL)a

Nanofectin FuGENE diluentb (µL)c HD (µL)

Opti-MEM/ D-MEM (µL)

Four- or 24-well MatTek Petri dish, 35-mm Petri dish, 10-cm

~2.5 ~1.5 ~2.0 ~4.0

50 100 100 250

50 200 200 500

0.17 1.0 1.3 3.3

0.5 3 4 10

0.75-1.5 3-6 3-6 20

2 8 8 20

a

Total volume = 1:3 dilution of cell suspension obtained from dish of 100% confluent cells. 150 mM NaCl; provided by manufacturer. c The final ratio of DNA to Nanofectin should be 1:3.2. b

16. Prepare the transfectant:

With Nanofectin: i.

For each dish or well, dilute the DNA such that the ratio of DNA to Nanofectin in the final mix (see Step 16.iii) is 1:3.2.

The amount and concentration of DNA will depend on the size of the dish and the number of cells (Table 1). ii. In a second tube, mix the Nanofectin with its diluent (provided by the manufacturer). iii. Add the Nanofectin solution to the DNA solution all at once. Mix immediately by tapping

or vortexing the solution. Do not mix in the reverse order. iv. Centrifuge briefly. v. Incubate for 15-30 min at room temperature.

With FuGENE HD: vi. For each dish or well, mix the DNA with D-MEM or Opti-MEM. The amount of DNA and the medium used will depend on the size of the dish and the number of cells (Table 1). The medium used should not include additives. vii. Add FuGENE HD to the DNA (Table 1). Vortex briefly. viii. Incubate for 20 min at room temperature. 17. Add the transfection mixture (from Step 16.v or Step 16.viii) dropwise to the prepared cells. Mix

gently. 18. Incubate for 4-24 h for downstream applications. Procedures are presented for observation of live cells (Steps 19-21), immunocytochemistry of fixed cells (Steps 22-33), and protein extraction for biochemical applications such as Western blotting or immunoprecipitation (Steps 34-40).

Observation of Live Cells 19. Culture cells in sterilized imaging chambers (Köster and Fraser 2004; Distel and Köster 2007) or

commercially available imaging chambers (e.g., MatTek). See Troubleshooting. 20. Transfect cells as described in Steps 14-18. 21. Observe live cells with an inverted microscope (Fig. 2). See Troubleshooting.

Immunocytochemistry of Fixed Cells 22. For each preparation, place a coverslip into the well of a four- or 24-well dish.


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FIGURE 2. Confocal image of a PAC2 cell transfected with Golgi (green) and cytosolic (red) DNA constructs. (Inset) Higher magnification of the Golgi apparatus. (For color figure, see doi: 10.1101/pdb.prot5235 online at www.cshprotocols.org.) 23. Plate cells on the coverslips using a 1:3 dilution of a suspension obtained from a dish of 100%

confluent cells. Use a total volume of 500 µL/well. Allow the cells to attach to the coverslips for at least 6 h at 28°C. See Troubleshooting. 24. Transfect cells using Nanofectin or FuGENE HD as described in Steps 14-18. Incubate overnight

at 28°C. 25. Wash cells once with PBS. 26. Fix cells in 4% paraformaldehyde for 10 min at 4°C. Do not extend the fixation step. 27. Wash the cells three times with PBS. Cells can be stored for ~1 wk at 2°C-8°C. 28. Prepare a blocking solution of PBS containing 10% NGS, 0.1% Tween 20, and 1% DMSO.

Incubate the cells with 300-500 µL of blocking solution for 30-60 min. 29. Incubate the cells with primary antibody against the protein or epitope of interest diluted in

blocking solution for 2 h at room temperature (or overnight at 4°C). 30. Wash the cells three times with PBS. 31. Incubate the cells with secondary antibody (targeted against the primary antibody) diluted in

blocking solution for 30 min at room temperature. 32. Wash the cells three times with PBS. 33. Mount the cells with an appropriate mounting medium. See Troubleshooting.

Protein Extraction This procedure is suitable for Western blot analyses, immunoprecipitation, or other biochemical applications. The example below is for a 10-cm dish. Adjust the volumes accordingly for other applications. 34. Wash a 10-cm Petri dish of 100% confluent cells once with PBS. 35. Add 0.8 mL of TEN to the dish. Place the cells on ice. Keep the samples on ice during cell lysis. 36. Use a cell scraper to remove the cells from the plate. Transfer the suspension to a 1.5-mL

microcentrifuge tube.


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37. Centrifuge at maximum speed for 10 min at 4°C. Remove the supernatant. 38. Resuspend the pellet in 100-1000 µL of IPN150. Incubate for 30 min at 4°C. 39. Centrifuge at maximum speed for 10 min at 4°C. Transfer the supernatant to a new tube. Reserve the pellet for analysis of the membrane/organelle fraction. 40. Store lysates for 24 h on ice (i.e., at 4°C). For longer storage, freeze lysates quickly in liquid nitrogen. Store frozen stocks at -20°C or -80°C.

TROUBLESHOOTING Problem: The cells take a very long time to detach or do not detach at all. [Step 2.ii] Solution: Consider the following: 1. Trypsin has an optimal working temperature of ~37°C. Incubate the plate in a 37°C incubator for

a short period (e.g., ~5 min). 2. Replace the trypsin-EDTA solution. Problem: Cells detach from glass surfaces. [Steps 19 or 23] Solution: Coating the surfaces onto which the cells are plated allows them to attach more strongly.

Coat surfaces as follows: 1. Prepare a solution of poly-DL-ornithine hydrobromide (mol. wt. 3000-15,000; Sigma P8638)

at 3 µg/mL in H2O. 2. Add the solution to the surface onto which the cells will be plated. Incubate for 30 min at room

temperature and then remove the coating solution. 3. Allow the coated surface to dry for at least 30 min. 4. When coating coverslips in a four- or 24-well dish, make sure that they are covered with the

solution (i.e., not floating on top). Coated dishes can be stored for up to 1 wk at 2°C-8°C. Problem: Transfection efficiency is very low. [Steps 21 or 33] Solution: Consider the following: 1. Monitor cell density. Only transfect cells when they are 50%-80% confluent. 2. Adjust the DNA concentration and/or the amount of transfection reagent. 3. Transfection efficiencies are usually sufficient using the methods described. If higher efficiencies are

required, nucleofection (He et al. 2006) can be used.

DISCUSSION Zebrafish PAC2 fibroblast cells are very easy to maintain and rarely overgrow. Because they grow at temperatures ranging from 20°C to 32°C, live cells can be imaged at room temperature for long periods. Furthermore, because PAC2 cells do not require a CO2-enriched environment, there is no need for additional specialized microscope equipment. The fact that they strongly attach to glass (e.g., coverslips) makes them suitable for immunohistochemistry. PAC2 cells proliferate rather slowly, and the cells are quite small. Nevertheless, they can be used for biochemical applications, such as antigen detection on Western blots or immunoprecipitation. The cells simply require enough time to grow and produce a reasonable amount of protein.

ACKNOWLEDGMENTS We thank Martin Distel, Jennifer Hocking, and Kazuhiko Namikawa for careful reading of the manuscript and discussion. This work was supported by the Ministry of Education and Research through a Biofuture Award Grant (0311889). www.cshprotocols.org

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REFERENCES Massachusetts Institute of Technology, Cambridge, MA. Distel M, Köster RW. 2007. In vivo time-lapse imaging of zebrafish embryonic development. Cold Spring Harb Protoc doi: 10.1101/ pdb.prot4816. He S, Salas-Vidal E, Rueb S, Gabby Krens SF, Meijer AH, Snaar-Jagalska BE, Spaink HP. 2006. Genetic and transcriptome characterization of model zebrafish cell lines. Zebrafish 3: 441–453. Köster RW, Fraser SE. 2004. Time-lapse microscopy of brain development. Methods Cell Biol 76: 207–235.

Amsterdam A, Burgess S, Golling G, Chen W, Sun Z, Townsend K, Farrington S, Haldi M, Hopkins N. 1999. A large-scale insertional mutagenesis screen in zebrafish. Genes & Dev 13: 2713–2724. Chen W, Burgess S, Golling G, Amsterdam A, Hopkins N. 2002. Highthroughput selection of retrovirus producer cell lines leads to markedly improved efficiency of germ line-transmissible insertions in zebra fish. J Virol 76: 2192–2198. Culp P. 1994. Random DNA integrations as an approach to insertional mutagenesis in the zebrafish (Brachydanio rerio). PhD thesis,


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Appendix 1: Recipes [NOTE: This print edition of CSH Protocols contains only recipes for reagents requiring multiple components or nonobvious critical steps. Recipes for reagents marked with the symbol not listed below can be found online at http://www.cshprotocols.org/recipes.]

Alkaline phosphatase buffer (APS) 50 mM MgCl2 100 mM NaCl 100 mM Tris, pH 9.5 0.1% Tween 20

AP buffer for honeybee 100 mM Tris-Cl (pH 9.5)

100 mM NaCl 50 mM MgCl2

Carbonate buffer for probe preparation 120 mM Na2CO3 80 mM NaHCO3 Adjust pH to 10.2.

Carnoy’s fixative Methanol, three parts Acetic acid (glacial), one part Prepare fresh for each use.

Denhardt’s reagent (50X) Reagent

Amount to add

Final concentration (50X)

Ficoll 400 Polyvinylpyrrolidone (PVP) Bovine serum albumin (Fraction V) H2O

1g 1g 1g to 100 mL (final volume)

1% (w/v) 1% (w/v) 1% (w/v) —

Dissolve the components in the H2O. Filter to sterilize and remove particulate matter. Divide into aliquots and store at –20°C. Usually, the 50X stock solution is diluted 10-fold into prehybridization buffer (e.g., 6X SSC or 6X SSPE containing 1.0% SDS and 100 µg/mL denatured salmon sperm DNA).

EDTA To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA•2H2O to 800 mL of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (~20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to ~8.0 by the addition of NaOH.


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ES-DMEM DMEM (high-glucose, 4500 mg/L) can be purchased as a powder or ready-made (e.g., Invitrogen). If it is reconstituted, use water free of bacterial endotoxins. Before use, supplement with: Glutamine to 2 mM from a 100X stock (200 mM, e.g., Invitrogen), stored as aliquots at −20°C. MEM nonessential amino acids to 0.1 mM from a 100X stock (10 mM, e.g., Invitrogen), stored at 4°C. 0.1 mM β-mercaptoethanol or 0.15 mM monothioglycerol (both tissue culture grade, Sigma), stored as aliquots at −20°C. (Optional) Sodium pyruvate to 1 mM from 100X stock (e.g., Invitrogen), stored at 4°C. Gentamicin to 50 µg/mL (50 mg/mL stock, Sigma), or penicillin to 50 U/mL and streptomycin to 50 µg/mL, or no antibiotics, particularly if cells are to be tested for Mycoplasma. A 100X stock of pen-strep (e.g., Invitrogen) is stored at −20°C. 15% fetal bovine serum (FBS) (e.g., Invitrogen, Sigma). Identify a batch that gives optimal growth and reserve a supply if possible. Leukemia inhibitory factor (LIF), 500-1000 U/mL for MEF feeders and 1000 U/mL for STO feeders (ESGRO/Millipore). Store in the dark at 4°C. If complete medium is stored for >2 wk, supplement with additional 1-2 mM L-glutamine.

FA lysis buffer EDTA (1 mM, pH 8.0)

HEPES-KOH (50 mM, pH 7.5) NaCl (140 mM) Protease inhibitors (100X), diluted to 1X final concentration Sodium deoxycholate (0.1%, w/v) Triton X-100 (1%, v/v)

FA500 buffer EDTA (1 mM, pH 8.0)

HEPES-KOH (50 mM, pH 7.5) NaCl (500 mM) Sodium deoxycholate (0.1%, w/v) Triton X-100 (1%, v/v)

Forensic DNA extraction buffer EDTA (50 mM, pH 8.0)

NaCl (100 mM) SDS (sodium dodecyl sulfate) (0.5%) Tris (10 mM, pH 8.0)

HEPES-buffered saline for lentivirus transduction (HBS-LT; 2X) Reagent

Amount to add

Final concentration

HEPES 2.38 g 0.05 M NaCl 3.28 g 0.28 M Na2HPO4 42.6 mg 1.5 mM Dissolve the ingredients in 200 mL of H2O. Sterilize by passage through a 0.2-µm filter.


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Hybridization buffer for honeybee 50% formamide 4X SSC 1X Denhardt’s reagent 250 µg/mL tRNA (or total yeast RNA) 250 µg/mL ssDNA 50 µg/mL heparin 0.1% Tween 20 5% dextran sulfate

Internal recording solution 10 mM HEPES 0.3 mM GTP 5 mM KCl 130 mM KMeO4 2.5 mM Mg-ATP 5 mM NaCl Adjust pH to 7.3.

Intra-pipette solution for WC recording 10 mM HEPES 4 mM KCl 4 mM MgATP 0.3 mM Na3GTP 10 mM phosphocreatine 135 mM potassium gluconate Adjust pH to 7.2 with KOH.

IPN150 MgCl2 (5 mM) NaCl (150 mM) Nonidet P-40 (0.1%, v/v) Tris-Cl (50 mM; pH 7.6) Immediately before use, add: DTT (Dithiothreitol) (1 mM) Phenylmethylsulfonyl fluoride (1 mM) Protease inhibitor cocktail, complete (1X; Roche Applied Science)

LiCl buffer for ChIP 0.25 M LiCl 0.5% Nonidet P-40 (NP-40) 0.5% sodium deoxycholate


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LB (Luria-Bertani) liquid medium Reagent

Amount to add

H2O Tryptone NaCl Yeast extract

950 mL 10 g 10 g 5g

Combine the reagents and shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (~0.2 mL). Adjust the final volume of the solution to 1 L with H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. For solid medium, see the recipe entitled “Media containing agar or agarose.”

LiCl wash EDTA (1 mM, pH 8.0)

LiCl (250 mM) Nonidet P-40 (0.5%, v/v) Sodium deoxycholate (0.1%, w/v) Tris-Cl (10 mM, pH 8.0)

Media containing agar or agarose Prepare liquid media according to the recipe given. Just before autoclaving, add one of the following: Bacto agar (for plates) Bacto agar (for top agar) Agarose (for plates) Agarose (for top agarose)

15 g/L 7 g/L 15 g/L 7 g/L

Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. When the medium is removed from the autoclave, swirl it gently to distribute the melted agar or agarose evenly throughout the solution. Be careful! The fluid may be superheated and may boil over when swirled. Before adding thermolabile substances (e.g., antibiotics), allow the medium to cool to 50°C-60°C, and mix the medium by swirling to avoid producing air bubbles. Before pouring the plates, set up a color code (e.g., two red stripes for LB-ampicillin plates; one black stripe for LB plates, etc.), and mark the edges of the plates with the appropriate colored markers. Pour plates directly from the flask; allow ~30-35 mL of medium per 90-mm plate. To remove bubbles from the medium in the plate, flame the surface of the medium with a Bunsen burner before the agar or agarose hardens. When the medium has hardened completely, invert the plates and store them at 4°C until needed. The plates should be removed from storage 1-2 h before they are used. If the plates are fresh, they will “sweat” when incubated at 37°C. When this condensation drops on the agar/agarose surface, it allows bacterial colonies or bacteriophage plaques to spread and increases the chances of cross-contamination. This problem can be avoided by wiping off the condensation from the lids of the plates and then incubating the plates for several hours at 37°C in an inverted position before they are used. Alternatively, remove the liquid by shaking the lid with a single, quick motion. To minimize the possibility of contamination, hold the open plate in an inverted position while removing the liquid from the lid.

MNase digestion buffer for ChIP 50 mM Tris-Cl (pH 7.6) 1 mM CaCl2 0.2% Triton X-100 5 mM sodium butyrate 1X protease inhibitor cocktail for ChIP 0.5 mM phenylmethylsulfonyl fluoride (PMSF), freshly prepared Nonidet P-40 (NP-40) can be used in place of Triton X-100.


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MNase stop buffer for ChIP 10 mM Tris-Cl (pH 7.6) 10 mM EDTA

NT buffer 100 mM Tris-Cl (pH 9.5)

100 mM NaCl

PAC2 medium Leibovitz’s L-15 medium, liquid, with L-glutamine (1X; GIBCO/Invitrogen) Fetal bovine serum, EU-approved (FBS; e.g., GIBCO/Invitrogen or PAA A15-101) MEM nonessential amino acids, without L-glutamine, liquid (100X; GIBCO/Invitrogen) Penicillin-streptomycin, liquid (GIBCO/Invitrogen)

Phosphate-buffered saline (PBS) Reagent

Amount to add (for 1X solution)


Amount to add (for 10X stock)


NaCl KCl Na2HPO4 KH2PO4

8g 0.2 g 1.44 g 0.24 g

137 mM 2.7 mM 10 mM 1.8 mM

80 g 2g 14.4 g 2.4 g

1.37 M 27 mM 100 mM 18 mM

If necessary, PBS may be supplemented with the following: CaCl2•2H2O 0.133 g 1 mM 1.33 g 10 mM MgCl2•6H2O 0.10 g 0.5 mM 1.0 g 5 mM PBS can be made as a 1X solution or as a 10X stock. To prepare 1 L of either 1X or 10X PBS, dissolve the reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.

Protease inhibitor cocktail for ChIP (1000X) Leupeptin (10 mg/mL) Chymostatin (10 mg/mL) Pepstatin A (10 mg/mL) Antipain (5 mg/mL) To prepare 1000X stock solution, dissolve the above protease inhibitors in DMSO (dimethyl sulfoxide).

Protease inhibitors (100X) Benzamidine (1.65 mg) Leupeptin (1.42 mg) Pepstatin A (6.85 mg) Phenylmethylsulfonyl fluoride (PMSF) (0.85 mg) Ethanol (50 mL) Dissolve the first four reagents in the ethanol. Store at −20°C.


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PTW buffer Tween 20 PBS Dissolve 0.1% (v/v) Tween 20 in PBS.

Ringer’s solution for VSD imaging 1.8 mM CaCl2 5 mM HEPES 5 mM KCl 1 mM MgCl2 135 mM NaCl

RIP elution buffer EDTA (10 mM)

SDS (sodium dodecyl sulfate) (1%) Tris-Cl (100 mM, pH 8.0)

RIPA buffer for ChIP 10 mM Tris-Cl (pH 7.6) 1 mM EDTA

0.1% SDS (sodium dodecyl sulfate) 0.1% sodium deoxycholate 1% Triton X-100

SSC For a 20X solution: Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 mL of H2O. Adjust the pH to 7.0 with a few drops of a 14 N solution of HCl. Adjust the volume to 1 L with H2O. Dispense into aliquots. Sterilize by autoclaving. The final concentrations of the ingredients are 3.0 M NaCl and 0.3 M sodium citrate.

STE 10 mM Tris-Cl (pH 8.0)

0.1 M NaCl 1 mM EDTA (pH 8.0) Sterilize by autoclaving for 15 min at 15 psi (1.05 kg/cm2) on liquid cycle. Store the sterile solution at 4°C.

TE buffer (pH 8) 100 mM Tris-Cl (pH 8.0) 10 mM EDTA (pH 8.0)


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TEN EDTA (1 mM)

NaCl (150 mM) Tris-Cl (40 mM; pH 7.6)

Tris-buffered saline (TBS) (1X) 50 mM Tris-Cl, pH 7.5 150 mM NaCl To prepare, dissolve 6.05 g Tris and 8.76 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.

Tris-buffered saline (TBS) for RIP NaCl (140 mM) Tris-Cl (10 mM, pH 7.5)

Tris-Cl Tris base HCl To prepare a 1 M solution, dissolve 121.1 g of Tris base in 800 mL of H2O. Adjust the pH to the desired value by adding concentrated HCl. pH

HCl

7.4 7.6 8.0

70 mL 60 mL 42 mL

Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 L with H2O. Dispense into aliquots and sterilize by autoclaving. If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature-dependent and decreases ~0.03 pH units for each 1°C increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5°C, 25°C, and 37°C, respectively.

Wash buffer for honeybee 50% formamide 2X SSC 0.1% Tween 20


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