1. Ca2+ and Mg2+ reduce the effectiveness of cell detachment from culture dishes and cause detached cells to clump.

2. NUNC dishes have thin walls and low intrinsic fluorescence, which are important advantages for the immunocytochemistry protocol described herein. If necessary, coating these dishes with poly-L-lysine or poly-L-arginine (Sigma) will improve the adhesion of trans-fected cells to the dish but may increase the background staining somewhat. To coat dishes, dissolve either amino acid at 5 |g/mL in water and filter sterilize (0.45 |im filter). This solution is stable at 4°C for 1 yr. Dilute this stock 100-fold in PBS (50 |g/mL, stable at 4°C for 1 yr) and add 1 mL to each culture dish. After 3 h, rinse each dish with 3 x 2 mL of PBS. Coated dishes can be stored sterile for 1 mo at 4°C.

3. Place several wet paper towels into a 150 mm culture dish with a lid. Each chamber accommodates up to 6 x 35 mm dishes and will remain moist overnight at RT.

4. To maximize fluorescence signals from labeled cells, the pH of this solution is very important. DABCO is alkaline in solution, so the DTG should be approx pH 8.6 after the addition of Tris. The pH can be easily measured after diluting the DTG 10-fold in distilled water.

5. Enzyme volume should be no more than 10% of the total.

6. Use protective gloves when handling the hot agarose solution, which can superheat and boil over, causing serious burns.

7. The known concentration of the ladder is used to estimate the concentrations of the samples by comparing band intensities under UV illumination.

8. Use 20-50 ng of plasmid DNA for the ligation. Increasing the molar ratio to more than 1:5 may cause 2 or more copies of the cDNA to be ligated into the pUC19 vector.

9. The 16°C incubation slows the enzyme activity and, theoretically, increases the ligation efficiency. However, for routine cloning the shorter ligation times yield sufficient numbers of bacterial colonies.

10. Do not use LB-ampicillin in this step because the bacteria need time to transcribe and translate the P-lactamase gene on the introduced plasmid. The 1 h incubation period can be eliminated to reduce processing time, which will reduce the number of colonies on the plates by two- or three-fold.

11. Ampicillin is heat labile and it is important to ensure the correct temperature of the LB-agar, which takes 1-2 h to cool to 55°C with occasional swirling of the bottle. After adding the ampicillin, mix thoroughly by swirling. When pouring the agar plates, large and small bubbles will collect on the liquid surface. Burst the bubbles by flaming the plates with a blue-yellow Bunsen flame for a few s. 500 mL of LB-agar makes up to 40 plates and can be stored for up to 1 mo at 4°C. Discard plates if fungal contamination is present.

12. To make a spreader, use a Bunsen burner to make a right-angle bend in the tapered end of a 9-in. Pasteur pipet. Sterilize with ethanol and briefly flame.

13. Spreading more than 100 |L of bacteria onto a plate should be avoided. Reduce the volume by microfuging the bacterial suspension for 20 s, withdrawing excess supernate to 100 |L and resuspending the soft pellet with a pipet.

14. The 4°C incubation increases the intensity of blue colonies. If the concentration of ampicillin in the plates is low, or if the transformed bacteria grow vigorously, true transformants will be ringed by small satellite colonies. Avoid picking the satellite colonies, which do not harbor plasmid DNA and will not grow in LB-ampicillin medium. In general, the longer the time of growth on the plates, the greater the number of satellite colonies. Thus, avoid growing transformants for longer times than is necessary to obtain 0.5-1 mm diameter colonies.

15. Colonies should be large, round, smooth and well-isolated.

16. The DNA concentration will be approx 0.1-0.2 |g/|L.

17. Although small, there is a finite risk of introducing point mutations into the cDNA during cloning and culturing the bacteria. Such mutations could confound all future work with the plasmid. Thus, the cDNA should be sequenced to ensure that random mutations are absent.

18. Include the PCR template DNA as a positive control. Correctly mutagenized plasmids will harbor one less Apa I site than the parent plasmid.

19. To achieve very high expression levels, pCMV5 is an ideal vector because it harbors an SV-40 origin of replication which the SV-40 T antigen expressed by COS-7 cells uses to replicate the plasmid to high copy number.

20. Use 70% ethanol to sterilize the surfaces of the culture hood, benches, Gilson pipets and outer surfaces of the culture-medium bottle. Allow the ethanol to evaporate.

21. Strike the side of the flask several times, which dislodges the cells when trypsinizing is complete.

22. Gently pipet cells up-and-down 5-10 times with a 10 mL pipet to break up cell clumps.

23. Growth time is generally 3-4 d if the cells are healthy. Slow growing cells may be due to a viral infection (e.g., mycoplasma), which will make the cells more difficult to transfect and will yield cells with poor morphology under the microscope. Discard the cultures and obtain a fresh flask of cells.

24. Cell number will approx double by d 2. 1 x T 75 flask at 100% confluence on d 1 yields approx 8 x 60 mm dishes at 50% confluence on d 2. Transfections also can be performed directly in individual 35-mm dishes rather than using a 60 mm dish intermediate. A disadvantage of this approach is that replicate dishes may have greater variability due to differences in transfection efficiencies. However, an advantage is that transfection times can be reduced to 15 h before fixation and staining which will yield higher apparent transfection efficiencies if the mutant protein being expressed is particularly toxic to the cells. We have used this approach to examine trafficking of a PLP1 splice site mutation (19) that results in skipping exon 6.

25. The FUGENE 6 must be added directly into the DMEM, not down the wall of the tube.

26. Gently move the dishes several times from side-to-side to evenly distribute the DNA on the cells. Be careful not to move dishes in a circular motion, which will concentrate reagents in the centers of the dishes.

27. The number of dishes prepared depends on the number of DNAs for transfection and the number of replicate dishes for each DNA. Each 60 mm dish will provide sufficient cells for up to 8 x 35 mm dishes.

28. The levels of confluence in each 60 mm dish should approximate 100% if the transfected plasmid is not toxic to the cells. However, proportionately less medium should be used for subconfluent dishes (e.g., use 2 mL of medium for cells at 50% confluence).

29. Gently move the dishes from side-to-side to evenly distribute the cells. The desired level of confluence when the cells adhere is 20-30%. After overnight culturing, a confluence of 40-50% is expected, which provides sufficient transfected cells to examine and plenty of room for the cells to flatten out for good morphology.

30. Keep 35 mm dishes on a rocking platform (e.g., Bellco, NJ, USA) during staining. Warm DMEM and fixative minimize changes to cell morphology before the cells are fixed. Add solutions to the edge of each dish to minimize cell damage. Use a low vacuum to hasten solution changes.

31. Detergents such as Triton X-100 and other permeabilizing agents such as methanol can extract some proteins, such as PLP1, from the membranes of fixed cells, which reduces the staining intensity.

32. Attach a wide-mouth P200 or P1000 Gilson pipette tip to a vacuum line. It is most important that all cells and liquid be removed in preparation for applying the PAP pen. These pens are very expensive and are easily damaged by contact with debris and aqueous solutions.

33. This and subsequent steps use small volumes of antibody-containing solutions to label antigens in transfected cells spread over a wide area. Therefore, it is important to ensure that the culture dishes are level. If antibody solutions are in plentiful supply, larger volumes, e.g., 100 or 200 |L can be used.

34. In this step, the DNA-binding fluor, 1 |g/mL 4,6, Diamidino-2-phenylindole (DAPI, Sigma, MO, USA), can be included for visualizing the nuclei. This compound strongly labels nuclei and aids the location of cells that are unlabeled or weakly labeled by antibodies. Some antigens are expressed at sufficiently low levels to require signal amplification using a biotinylated secondary antibody and a streptavidin-conjugated fluorophore. Simply repeat this step with the necessary tertiary reagents.

35. Colored nail polish contains compounds that quench the fluorescence and can bleed into the DTG with time.

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