Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

Plating CHO-K1 Cells:

1. Add 50-100μl of CHO-K1 cells that have been trypsinized according to standard protocols to 6ml of complete F-12K.
2.  Mix cell suspension by pipetting up and down several times, then aliquot 300 μl of trypsinized cell suspension to each well of a sterile 8-chamber Lab-Tek II Chambered Coverglass with #1.5 borosilicate coverglass chambers (Nalgene Nunc Int.; Product #155409; Lot: 100808-8-0).
3. Incubate samples at 37°C, 5% CO₂ overnight.

Transient Transfection of CHO-K1 Cells with pSNAP-ADRB2 and pSNAP-H2B:

1. Check the cell culture chambers seeded on the previous day to look for cell density and health.
2. Label the appropriate number of microfuge tubes.
3. In a laminar flow hood, set up transfection complex mixtures according to the following sample chart, using either 0.3 μg pSNAP-ADBR2 Control Plasmid DNA or 0.3 μg of pSNAP-H2B Control Plasmid DNA, 1 μl of TransPass D2 and 1 μl of TransPass V per reaction.

   plasmid	number of reactions	μl of serum-free medium	μl of TransPass D2	μl of TransPass V	μl of plasmid DNA	μl of TransPass D2/reaction	μl of TransPass V/reaction	ng of DNA/ reaction	DNA conc. (ng/μl)	μl of plasmid DNA/ reaction
   SNAP-ADRb2	5	236.5	5	5	3.5	1	1	300	472.34	0.7
   H2B-SNAP	5	237	5	5	3	1	1	300	515.89	0.6
   Mock	5	240	5	5	0	1	1	0	0	0
   Untransfected	5	250	0	0	0	0	0	0	0	0

4. First, mix the serum free F-12K with the appropriate plasmid DNA, then add the TransPass D2 Transfection Reagent, then add the TransPass V Transfection Reagent. Mix the components well by pipetting up and down gently several times after the addition of each reagent.
5. Incubate the reaction at room temperature for 30 minutes.
6. During the incubation, wash cells once with 600 μl of complete DMEM and add 450 μl of complete DMEM to each sample.
7. Add 50 μl of the transfection complex mixture to each sample, slowly and in a drop-wise fashion.
8. Incubate the samples overnight at 37°C, 5% CO₂.

SNAP-Cell TMR Star Labeling of CHO-K1 Cells Transiently Transfected with pSNAP-ADRB2 and pSNAP-H2B :

1. Carefully remove the transfection complex media from each sample by vacuum suction. Wash cells twice with 600 μl of complete F-12K and replace media in each well with 600 μl of complete F-12K.
2. Mix dye labeling media by adding 995 μl of complete F-12K and 51 μl 0.6 mM SNAP-Cell TMR-Star  substrate. The final concentration of SNAP-Cell TMR-Star should be 3mM.  Pipette up and down several times to mix after the addition of substrate.
3. Carefully remove growth media from samples and add 200 μl of dye labeling media to each well. Incubate samples at 37°C, 5% CO₂ for 30 minutes.
4. While samples are incubating, prepare Hoechst 33342 solution for nuclear staining of samples by mixing 1 μl of Hoechst 33342, trihydrochloride, trihydrate (10 mg/ml solution in water, 16.2 mM solution; Invitrogen Molecular Probes) in 10 ml of complete F-12K.
5. Remove the media from all samples and add 600 μl of the dilute Hoechst solution to each sample. Incubate samples at 37°C, 5% CO₂ for 5 minutes.
6. Wash samples three times with 600 μl of complete F-12K.
7. Incubate the samples at 37°C, 5% CO₂ for an additional 30 minutes to allow unincorporated fluorophore to diffuse out of the cells.
8. After 30 minutes, change the media on the SNAP-Cell samples one last time and proceed to imaging.
9. Image the cells using a Zeiss Apotome Axiovert 200M fluorescent microscope.

Representative Results

Figure 1. Here are some representative results of fluorescent imaging using standard fluorescent microscopy of live COS-7 cells that are expressing SNAP-tag fusions. This first image shows live COS-7 cells expressing pSNAP-H2B (nuclear histone) labeled with SNAP-Cell TMR-Star. The pSNAP-H2B construct was generated using the pSNAP-tag(m) vector. The cells were labeled with SNAP-Cell TMR-Star for 30 minutes and counterstained with Hoechst 33342 (blue) for nuclei. The pink fluorescence demonstrates the overlay of red fluorescence where histone protein is present in addition to the blue fluorescence indicating the nucleus. The expression of the H2B protein is clearly and easily identified.

Figures 2 and 3. These next two images show live COS-7 cells transiently transfected with pSNAP-ADRβ2.
Cells were labeled with SNAP-Cell TMR-Star (red) and counterstained with Hoechst 33342 (blue). The pSNAP-ADRβ2 construct was generated using the pSNAP-tag(m) vector. The cells were labeled with SNAP-Cell TMR-Star for 30 minutes and counterstained with Hoechst 33342 (blue) for nuclei. The red fluorescence indicates the presence of cell surface receptor protein ADRB2 while blue fluorescence identifies the nucleus.