CellRapeutics™ Flow Cytometry Kit
Conventional methods for analyzing cell surface markers on adherent cells by flow cytometry require detachment of cells from their culture surface before antibody staining. Unfortunately, cell detachment introduces a significant stress to cells, which can lead to changes in expression of cell surface markers. In addition, multiple centrifugation step often are required during antibody staining and washing, which are not only laborious, but also can lead to cell loss. The CellRapeutics™ flow cytometry kit offered by Biotium provides a much simpler, more accurate and often more sensitive method for detecting cell surface markers on adherent cells than conventional methods. The CellRapeutics™ method eliminates laborious centrifugation steps. More importantly, CellRapeutics™ allows you to detect cell surface marker expression on cells in their native adherent state without perturbation caused by cell detachment. The CellRapeutics™ method can yield substantially higher fluorescence signal for cell surface markers compared to conventional methods.
Stable for at least 6 months from date of receipt if stored as directed.
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Reagent and instrument requirements
Antibody diluent (e.g. PBS + 1% BSA)
1. Seed cells onto a 24 or a 48 well plate and grow them to 75-90% confluence for the assay. Be sure to seed enough wells for samples and controls (1 assay/well). See Helpful Tips for cell seeding guidance.
2. Prepare the antibody(s) to the appropriate concentration normally used in a flow cytometry experiment. For each 48-well, 100 uL antibody solution is used. For each 24-well, 200 uL is used. Place the antibody solution on ice.
3. Take the plate out of the incubator. a. For unlabeled primary antibody, chill the plate on ice. b. For labeled primary antibody, leave the plate at room temperature.
4. Aspirate media and add 100 uL cold antibody solution into each 48 well or 200 uL into each 24 well.
5. Incubate on a rocker for the appropriate amount of time required for antibody binding. See Helpful Tips for recommendations. a. For unlabeled primary antibody, incubate at 4o C or on ice to avoid internalization of the receptor-antibody complex. b. For labeled primary antibody, incubate at room temperature. Then skip to Step 10. 6. During the primary antibody incubation, prepare the fluorescent-labeled secondary antibody conjugate at the appropriate concentration based on manufacturer's recommendation. Place on ice.
7. After incubation, aspirate the antibody solution and rinse 1X with 1 mL ice cold PBS.
8. Add 100 uL (48 well) or 200 uL (24 well) of fluorescent-labeled secondary antibody conjugate.
9. Incubate 30-60 minutes on a rocker at 4o C or on ice. During this time, warm Cell Lift Solution to 37o C.
10. Aspirate the labeled antibody solution and wash with 1 mL 1X PBS.
11. To each well add 100 uL (48 well) or 200 uL (24 well) Cell Lift Solution.
12. Incubate at 37o C for 10 minutes. Observe by microscopy to see if cells have been detached from each other. If not, continue 37o C incubation for additional 10 to 20 minutes.
13. Place the plate on ice. a. For cells that are not strongly adherent, dissociate cells by pipetting the cells a few times in each well and transfer to flow cytometry tube. Place the tube on ice. b. For cells that are strongly adherent, scrape the cells from the bottom of the plate using a Mini Cell Scraper, then dissociate cells by pipetting the cells up and down and transfer flow cytometry tube. Place the tube on ice. See Helpful Tips for additional guidance on hard-to-dissociate cells.
14. Rinse the well with 100 uL Stabilization Solution and transfer to the corresponding flow cytometry tube on ice.
15. Analyze samples by flow cytometry within 2 hours. Alternatively, fix the samples as described in Helpful Tips and analyze the next day.
1. Typical seeding densities for cells to be assayed on next day are 0.8x105 cells per 48 well or 1.5x105 cells per 24 well. Seeding density may require optimization for different cell types.
2. For cells grown in serum free media, include 10% calf serum in the diluent for primary antibody staining to block Fc receptors on the cells.
3. When staining a full plate, perform each step one row at a time to prevent the cells from drying out after aspiration. 4. Antibody binding to cells in the adherent state is as efficient as binding to cells in the suspension state. Follow your regular protocol for antibody binding.
5. When pipetting cells to break up any remaining cell clumps, be sure to pipet each well the same number of times before transferring cells to flow cytometry tubes.
6. For highly adherent cells such as A431, you may want to use a 24-well plate for higher cell number. At Step 13, after scraping cells off the wells and dissociating by pipetting, transfer cell suspension into a microcentrifuge tube and run the bottom of the tube across a row of wells of an empty Styrofoam 15 mL conical tube rack to break up cell clumps. Then transfer cells to flow cytometry tubes.
7. If you do not plan to perform flow cytometry within 2 hours of staining, fix cells in formaldehyde overnight and perform flow cytometry the next day. To fix cells in 2% formalin, add ¼ volume of 10% formalin (4% formaldehyde in PBS) to the cell suspension after step 14. Alternatively, add an equal volume of Biotium's cell fixation buffer to the cell suspension after step 14. Store overnight at 4o C, protected from light.
For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.