FC PROTOCOL RBD labeling of live cells for flow cytometry analysis 1. Introduction Fluorescence intensity The RBD labeling protocol for flow cytometry has been developed for you to get results in an easy, robust way. This protocol is highly comparable to what you are familiar with, i.e. cell surface antibody labeling. However, there are some differences that we highlight in the text to let you identify critical steps. If you have any question before beginning to use the RBD, or during your experiment, please feel free to call or send us an email, our support team will be pleased to help you. support@metafora-biosystems.com / Phone +33 (0)1 60 87 89 25 (Paris time, GMT +2hrs.) 2. Before using your RBD 2.1. Designing the flow cytometry color panel This protocol is designed and optimized for cell surface labeling of nutrient transporters with RBD, on live cells. You may use a viability marker to exclude dead cells during the analysis. All steps are conducted on live cells but optional fixation of labeled samples with 0.1 % PFA can be done before acquisition. If you are using a Fc fusion RBD Labeling is achieved by a two-step procedure; the RBD is purified, so that you need to reveal it with a dye-coupled, secondary antibody. We recommend using the brightest dye available, such as R-PE, APC etc., depending on your flow cytometer configuration. You may also want to use the RBD along with other cell surface markers; in this case, take into account your RBD tag (either mouse or rabbit Fc), to avoid cross reaction between primary antibodies and the RBD targeting secondary antibody. If you are using the GFP fusion GLUT1.RBD Labeling is achieved by a single-step procedure, as this RBD behaves like a primary antibody. This RBD is concentrated, but not purified. This protocol describes a two-color panel, using only the RBD and its secondary antibody plus DAPI as a viability marker. But remember that the RBD is a quite versatile tool so you can include it into more complex panels. RBD labeling on live cells for flow cytometry analysis Page 1 out of 5
2.2. RBD thawing and aliquoting We recommend not to perform too many thawing/freezing cycles to preserve the RBD. Upon first use, we suggest that you aliquote the RBD in individual tubes, with volumes corresponding to the average experiment size you intend to perform. We adjust the RBD concentration of each lot for you to obtain binding saturation with 1 to 5 µl on main cell types. However, we highly recommend to titrate each batch of RBD on your cells for optimal quantification, before using the RBD to generate results. 2.3. Buffer preparation Prepare enough buffer according to the number of samples to be processed. Store buffers at 4 C during the labeling procedure. Complete culture medium (containing FCS) - The first RBD labeling step is performed in your complete cell culture medium. Buffer A: RBD labeling buffer - Complete culture medium - 0.09 % NaN 3-1 mm EDTA Reagents For 1 sample (µl) For 8 samples (µl) * For 96 samples (µl) * Complete culture medium 98 980 11760 5 % NaN 3 1.8 18 216 0.5 M EDTA 0.2 2 24 Final volume 100 1 000 12 000 *volumes are calculated for the corresponding sampling + 20% Buffer B: 2 nd step labeling / acquisition buffer - 1X PBS - 0.09 % NaN 3-1 mm EDTA - 2 % FCS Reagents For 1 sample (µl) For 8 samples (µl) * For 96 samples (ml) * 1X PBS 739.6 7396 88.8 5 % NaN 3 13.9 139 1.67 0.5 M EDTA 1.5 15 0.18 FCS 15 150 1.8 Final volume 770 7 700 92.5 *volumes are calculated for the corresponding sampling + 20% Washing buffer: use 1X PBS to wash the cells after first and second labelling steps. RBD labeling on live cells for flow cytometry analysis Page 2 out of 5
PROTOCOL «AT A GLANCE» FOR GLUT1.RBD 1. Fill two FACS tubes per sample with cells (100 µl per tube at 0.5 to 1 10 6 cells.ml -1 in buffer A, one tube for the RBD labeling, the second one for the background) 2. RBD labeling Add 1 to 5 µl of Glut1.RBD (according to the best dilution determined previously) to the first FACS tube, for each sample 3. Incubate for 20 min at 37 C; please respect strictly this first labeling step as RBDs bind weakly at 4 C If you are using the GFP-fused RBD, proceed directly to step 7, otherwise proceed with steps 4 to 6 4. Wash cells with 200 µl of 1X PBS, pellet cells by spinning at 400, 4 C and 4 minutes then carefully discard supernatant 5. Secondary labeling Add 100 µl of secondary antibody (anti-mousefc) diluted at the optimal dilution in buffer B to all tubes 6. Incubate at 4 C in the dark for 30 minutes 7. Wash cells twice with 200 µl of 1X PBS, pellet cells by spinning as previously 8. Acquisition - Resuspend cells in 100 µl of buffer B, cells are ready for flow cytometry acquisition 9. Data analysis - Once you have applied your gating strategy to all samples, you can perform all type of graphic and calculation analysis you are used to in flow cytometry experiments and cellular assays (histograms, overlays, heatmap) RBD labeling on live cells for flow cytometry analysis Page 3 out of 5
3. Cells preparation for flow cytometry 3.1. Prepare the RBD labeling buffer (Buffer A), 3.2. Remove the culture medium, 3.3. Rinse the cells twice with 1X PBS, 3.4. Detach cells with an enzymatic dissociation buffer, Nutrient transporters are not too sensitive to enzymatic dissociation buffers commonly used to detach cell monolayer; however, we recommend using the mildest buffer at your disposal. 3.5. If you use trypsin, incubate 3 to 5 minutes at 37 C, 5% CO 2, or the minimum time required to detach your cells, 3.6. Check cells detachment under the inverted phase-contrast microscope: the cells must be in suspension and appear round, 3.7. Stop the reaction with one volume of complete culture medium for one volume of enzymatic dissociation buffer, 3.8. Lift up the remaining cells by pipetting up and down against the inner wall of the flask, 3.9. Transfer the cells in a 15 or 50 ml centrifuge tube, 3.10. Determine viability and viable cell number by Trypan blue exclusion, 3.11. For each sample, prepare a suspension of cells at a concentration between 0.5x10 6 and 1x10 6 cells/ml in buffer A. Prepare enough cells for the intended number of RBD labelings, plus the negative control, 3.12. Transfer 100 µl (i.e. about 50 000 to 100 000 cells) of this cell suspension in polypropylene tubes (or in a well of a 96-well V-shape microplate). We highly recommend to perform RBD labeling on live cells, except if your experiment design requires cells to be fixed. Because the RBD is designed to quantify the nutrient transporter at the cell surface exclusively, do not permeabilize the cells before RBD labeling. 4. RBD labeling 4.1. Thaw the RBD aliquot(s) to be used on ice, 4.2. Once the RBD solution is thawed, mix by pipetting, 4.3. Then spin it for 10 seconds, just enough to spin down protein aggregates, 4.4. Add the optimal quantity of RBD to the 100 µl of cell suspension of each sample, We adjust the RBD concentration of each lot so that you obtain binding saturation with 1 to 5 µl on main cell types. However, we highly recommend to titrate each batch of RBD on your cells for optimal quantification, before using the RBD to generate results. 4.5. Briefly vortex the samples, 4.6. Incubate for 20 min at 37 C; please respect strictly this first labeling step as RBD binds weakly at 4 C. If you are using the GFP fusion GLUT1.RBD, proceed to part 7 (Washing). If you are using a Fc fusion RBD, proceed to parts 5 and 6 (washing and secondary labeling). RBD labeling on live cells for flow cytometry analysis Page 4 out of 5
5. Washing 5.1. Add 200 µl of cold 1X PBS, 5.2. Pellet the cells by spinning at 400 G for 4 minutes at 4 C, 5.3. Aspirate the supernatant, or discard it by turning the tubes (or microplate) over, 5.4. Resuspend the cell pellet in the residual liquid by vortexing. 6. Secondary labeling 6.1. Dilute your anti-mouse Fc secondary antibody in buffer B, according to the best dilution determined by the titration curve, 6.2. Add DAPI at 1 µg/ml (1/1 000 when starting with DAPI stock solution at 1 mg/ml), Caution : if you intend to fix your cells after labeling, do not use DAPI as this dye will enter your fixed cells, and no washing can get read of it (all your cells would be DAPI positive), 6.3. Incubate at 4 C in the dark for 30 minutes (you may use longer incubation time with no impact on your results). 7. Washing 7.1. Add 200 µl of cold 1X PBS. 7.2. Pellet the cells by spinning at 400 G for 4 minutes at 4 C. 7.3. Aspirate the supernatant, or discard it by turning the tubes (or microplate) over. 7.4. Resuspend the cell pellet in the residual liquid by vortexing. 7.5. Repeat steps 7.1 to 7.4 a second time. 8. Acquisition buffer 8.1. Add 100 µl of acquisition buffer (Buffer B) to the cell pellet, 8.2. Store the cells at 4 C protected from light, 8.3. Sample is ready for flow cytometry analysis, 8.4. Optional fixation - If the acquisition can t be performed the same day, cells and labelings can be stabilized with a mild fixation consisting in 0.1% PFA in buffer B, or fix your cells with 2% PFA for 10 minutes at room temperature before acquisition. 9. Gating strategy Here is an example of the gating strategy applicable for the analysis of cell surface nutrient transporter V1 Application date April 27 th, 2014 expression on Mesenchymal Stem Cells. Acquisition was performed with a 3-laser flow cytometer. 4- Analysis of cell surface nutrient transporter expression Count AF647 Fluorescence intensity RBD labeling on live cells for flow cytometry analysis Page 5 out of 5