FREQUENTLY ASKED QUESTIONS
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DO NOT FREEZE. Storage below 4°C will cause irreversible damage to the MagCloudz™ and will drastically reduce kit performance.
Should your cell separation require starting populations greater than 107 cells per sample, we recommend that you scale up to 50 mL tubes and follow our ‘large scale’ cell separation protocol found on our Resources page.
It is recommended that the purity of the final isolated population be quantified using flow cytometry. Isolated cells should be re-suspended in appropriate flow cytometry staining buffer (e.g. PBS plus 1% BSA or 2% FBS) and an aliquot (containing 105 cells) transferred to a clean tube or 96-well plate for flow cytometry staining. Standard protocols for flow cytometry staining should be used for the desired cell surface and viability markers for the starting, un-bound and isolated cell populations The isolated population should be compared against the starting population (Step 1) and un-bound cell population (supernatant from Step 4.2) to determine target cell uptake, purity and enrichment.
For best cell separation results, we strongly recommend that fresh preparations of cultured cells or blood samples be used with the MagCloudz™ Streptavidin kit; however, frozen cell samples may be used if fresh cells are unavailable. Due to the stressful nature of the freeze-thaw cycle, cell viability and kit performance may be reduced.
If you use a previously frozen cell sample, be sure to rinse the cells after thawing in media containing 10% FBS to remove the freezing media, followed by a rinse in our 1X Cell separation buffer. (If cell clumping occurs during the separation process, we recommend trying the additional cell strainer and DNAse incubation step detailed below for frozen PBMC samples.)
For frozen PBMC specifically, we recommend that you first strain the cells using a 0.77 µm cell strainer to remove any cell clumps. Next, incubate the strained cells with 0.1 mg/mL DNAse for 15 minutes at room temperature following the media rinse to minimize cell clumping during the separation protocol. After the incubation with DNAse, spin down and rinse with 1X Cell Separation Buffer and proceed with the rest of the protocol as written. A second straining step may be required if cells still appear clumpy after incubation with DNAse.
If you wish to evaluate the target cell uptake efficiency, the un-bound cells in the supernatant from Step 5.2 should be collected and analyzed via flow cytometry along with the starting population and isolated population samples. Uptake is defined as the target cell percentage (%) present in the starting population minus the target cell percentage (%) present un-bound fraction (Step 5.2 supernatant) divided by the starting population target cell percentage (%).
The ‘theoretical’ target cell population can be calculated by multiplying the total number of cells in the starting population by the percentage of target cells present in the starting population. Target cell recovery can then be calculated, by dividing the total number of target cells obtained in Step 6 by the ‘theoretical’ target cell population.
For A), we recommend that you run an isotype control sample using an appropriate biotinylated Ig-G to evaluate non-specific cell binding in your particular sample. If high levels of non-specific binding are observed, the number of wash steps should be increased until optimal purity is obtained for the target cell population.
For B), MagCloudz™-target cell complexes must be carefully washed 2 times in 1X Cell Separation Buffer in Step 4 of the protocol. Gently pipet the sample up and down 3-4 times using a 1 mL pipet tip to get rid of any non-specifically bound cells. As mentioned above, the number of wash steps may need to be optimized to obtain high purity of the target cell population.
Low target cell recovery may occur if MagCloudz™-target cell complexes are washed too vigorously in Step 5, which can cause shearing and loss of target cell populations. Be sure to pipet gently throughout the washing process.
Additionally, low target cell recovery may also occur if: A) Cell Separation Buffer is mistakenly added instead of Cell Release Buffer in Step 6 or B) MagCloudz are not pipetted enough during Step 6 and incomplete cell release occurs or C) the released target cell populations are mistakenly discarded during Step 6. Ensure that 1X Cell Release Buffer is added during Step 6 and that samples are pipetted vigorously (up and down 10 times minimum) to completely release the target cells from the MagCloudz. At this stage, your target cells will be present in the SUPERNATANT, DO NOT DISCARD. Be sure to transfer the supernatant from Steps 6.2 and 6.3 into a single clean tube and spin down to pool the isolated cell population.