FREQUENTLY ASKED QUESTIONS

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Cells from mammalian sources can be used with the MagCloudz™ Streptavidin kit. Cells of plant or insect origin have not been tested; optimization may be required.

Typical protocol times range from 2-3 hours depending upon the number of samples.

YES. The kit is designed to process up to 100 samples containing 10 million (107) cells each, for a total of 1 billion (109) processed per kit. Multiple samples can be processed at once depending on the number of positions in the magnetic stand. Q-Mag magnetic stand can accommodate processing a maximum of sixteen 1.5 mL -2.0ml  samples simultaneously.

All MagCloudz Streptavidin kits expire one year from the date of manufacture. Kits should be stored in the refrigerator at 4°C while not in use.

MagCloudz™ Streptavidin kits should be stored in the refrigerator at 4°C upon arrival. Diluted buffers and un-used MagCloudz™ may be kept at 4°C for up to 6 months after opening. Keep all kit components cold (on ice) during the protocol to maximize kit performance.

DO NOT FREEZE. Storage below 4°C will cause irreversible damage to the MagCloudz™ and will drastically reduce kit performance.

NO. Cells are completely released from the MagCloudz™ during the final steps of the protocol and no magnetic particles are retained on the cell surface.

YES. The MagCloudz Streptaviding kits can be used for positive selection of the desired target cell population OR for negative selection via depletion of undesired cell populations using a biotinylated antibody cocktail for lineage depletion.

YES. While the cells are released from the QuickGel and magnetic particles, the biotinylated antibody complexed with streptavidin remains on the cell surface.

We recommend that the separation protocol be carried out using 2 mL Lo-Bind Eppendorf tubes for samples containing up to 107 total cells. These are designed for minimal non-specific binding and work best with our MagCloudz™. However, standard 1.5 mL Eppendorf tubes may also be used for the separation protocol. Samples collected for flow cytometry staining and the isolated cell populations may be collected in your vessel of choice.

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.

The differences in magnetic field strength of various manufacturer’s magnetic stands can cause variation in the performance of the MagCloudz™ Streptavidin kit. We highly recommend using the Q-Mag magnetic stands, which has been validated to work optimally with the MagCloudz kit’s magnetic separation steps.  Other comparable magnetic stands may also be used, however  protocol optimization may be required to achieve full separation of the MagCloudz from the supernatant. 

YES. The presence of any free/un-bound biotnylated antibody in the cell sample will reduce the binding capacity of the MagCloudz and therefore decrease target cell uptake. We recommend a rinse in our Cell Separation Buffer to remove excess antibody prior to mixing the cells with the MagCloudz.

We recommend that experimental samples, stained with the desired biotinylated antibody, are run in triplicate. We also recommend that a negative control sample, stained with a biotinylated-Ig-G isotype antibody, is run along with the experimental samples to evaluate any non-specific binding to the MagCloudz in your particular cell type.

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.

If you do not have access to a cold room or refrigerator, the 30 min MagCloudz™ Streptavidin incubation (Step 3.3) may be performed on the lab bench at room temperature. However, be sure to keep all buffers and antibodies cold throughout the entire protocol for optimal kit performance.

YES. All MagCloudz™ Streptavidin kit components are manufactured in an aseptic environment and will remain aseptic until opened if handled correctly.
YES. If the protocol is followed precisely, isolated cells will be highly viable and suitable for subsequent culture and expansion. If post-separation culture is desired, be sure to maintain sterility of ALL kit components by handling the kit and cell samples in a cell culture hood.
A biotinylated antibody which specifically targets your cellular marker of choice should be selected for your given application. We recommend using antibodies which have been validated for specificity and sensitivity.

We recommend following the manufacturer’s directions for staining your target cells with biotinylated antibody. As with any other immuno-labeling application, titration and optimization may be required for maximum kit performance.
Cells may be cultured in any media prior to and/or after the separation protocol. If media formulations containing biotin or cell dissociation reagents containing chelating agents such as EDTA were used to prepare the cells prior to Step 2, be sure to rinse the cell sample 1-2 times in 1X Cell Separation Buffer for optimal kit performance. The presence of residual biotin, enzyme or EDTA-based reagents may reduce the binding capacity of the MagCloudz™ Streptavidin and lower target cell recovery.

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 are using whole blood samples to derive mononuclear cell fractions, we strongly recommend heparinized tubes for blood collection. Collection tubes containing EDTA are not recommended for use with this kit.
During the cell release process, certain components of the MagCloudz™ are ‘dissolved’, which alters the appearance of the magnetic particles collected on the tube wall against the magnet. Do not be alarmed! This is typical behavior and is an indicator that cell release has occurred.
We recommend that you analyze your starting population and isolated cell populations using phenotype and viability markers via flow cytometry. Standard protocols and manufacturer’s recommended staining instructions should be followed to prepare the cell samples for flow cytometry analysis.

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.

Low purity (<70%) of isolated cell populations may occur if: A) there are high levels of non-specific cell binding in your cell sample or B) if MagCloudz™-target cell complexes are not washed appropriately using Cell Separation Buffer in Step 5, prior to the release step.

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.

Our kit is designed for high yield recovery of target cell populations, averaging ≥ 60% recovery of the initial target cell population. If you experience low recovery (<50%), we recommend that you verify that the optimal biotinylated antibody staining protocol was followed to stain the desired target cells in Step 1 and that the antibody clone indeed binds to the intended 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.

Our technical specialists are available to help you. If you need additional technical support, please fill out our contact form or email us directly at info@quadtechnologies.com. We’ll be happy to help you!