Babco, Signet, Sternberger Monoclonals, Senetek, Covance

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Cell-Vive™ GMP MojoSort™ Human CD56 Nanobeads

480205

100 tests

1980 USD

Cell Sep - Pos

480205

Cell-Vive™ GMP MojoSort™ Human CD56 Nanobeads

100 tests

1980 USD

Human

CV_GMP_MOJO

This kit is optimized for the positive selection or depletion of CD56+ cells from human peripheral blood mononuclear cells (PBMCs). Alternate sources of cell suspensions may require protocol optimization.; Recommended protocol for positive selection or depletion of CD56+ cells from human PBMCs; Use sterile consumables and work in a biosafety cabinet. To maintain cell viability and reduce non-specific binding, work quickly, keep cells cold, and use pre-chilled separation buffer kept on ice throughout the procedure.; Isolate PBMCs using density gradient centrifugation. If working with another tissue, prepare a single cell suspension according to standard protocols. Excess dead or red blood cells may increase non-specific binding and removal prior to separation may increase performance. Filter the cell suspension with a sterile 40 µm nylon cell strainer for optimal performance.; Separator Protocol;This procedure is optimized at 1 x 10^7 cells/test. When working with more cells, optimize the conditions to your specific cell number and tissue for best results.;Resuspend cells at 1 x 10^8 cells/mL with Cell-Vive™ CD Cell Separation Buffer, GMP (Cat# 420512) or other commercially available separation buffer.;Aliquot 100 µL (10^7 cells) into appropriate tube.;Optional: Reserve an aliquot of unseparated cells as a control.;Scale up to 2 mL (2 x 10^8 cells) in 12 x 75 mm (5 mL) polypropylene tube for 5 mL magnet (Cat# 480019).;Scale up to 10 mL (1 x 10^9 cells) in 17 x 100 mm (14 mL) polypropylene tube for 14 mL magnet (Cat# 480020).;Resuspend the antibody-conjugated nanobeads well by vortexing at maximum speed, at least 5 touches. Add 10 µL of conjugated nanobeads per 10^7 cells. Immediately, mix well to fully incorporate, and incubate on ice for 15 minutes.;Optional: Washing cells to remove unbound nanobeads may improve purity but reduce yield. Use separation buffer, gently mix and centrifuge the cell suspension at 300xg for 5 minutes and discard supernatant.;Add separation buffer, up to indicated volume. Mix well by pipetting to fully incorporate.3.5 mL for separating ≤ 2 x 10^8 cells using the 5 mL magnet.;5 mL for separating ≤ 5 x 10^8 cells, or 10 mL for separating 5 x 10^8 - 1 x 10^9 cells, using the 14 mL magnet.;Place the tube in the MojoSort™ magnet to perform separation.5 minutes separation time for 5 mL magnet.;10 minutes separation time for 14 mL magnet.;Carefully, pour out the unlabeled fraction into a collection tube. If these are your cells of interest, do not discard. Remove tube containing magnetically labeled fraction from the magnet, do not discard.;Repeat steps 4-6 on the magnetically labeled fraction for a total of 3 separations for optimal purity. Pool the unlabeled negative fractions and keep the labeled cells for downstream applications. The fraction that is not of interest may be useful as staining controls, to monitor purity/yield, or other purposes.; Column Protocol;Prepare antibody-conjugated nanobeads at dilution recommended for column use found on the TDS. Vortex nanobeads well at maximum speed to resuspend before diluting with Cell-Vive™ CD Cell Separation Buffer, GMP (Cat# 420512) or other commercially available separation buffer.; Cat# 480204/480205 recomended nanobead dilution to use on columns: 6.5x; There are several types of commercially available columns, depending on your application. Choose the one that best fits your experiment. Column cell capacity is dependent on column volume, target cell abundance, type, and size. Protocol and bead dilution should be optimized empirically for use on different column systems.;Resuspend cells at 1 x 10^8 cells/mL with separation buffer.;Optional: Filter cell suspension through a 40 µm filter before addition to column for best results.;Aliquot 100 µL (10^7 cells) into appropriate tube.;Optional: Reserve an aliquot of unseparated cells as a control.;Note: Scale up volumes for larger cell numbers dependent on column capacity. For example, add 100 µL nanobeads for 1 x 10^8 cells (equivalent to 10 tests at 1 x 10^7 cells/test) in 1 mL separation buffer.;Resuspend the diluted antibody-conjugated nanobeads well by vortexing at maximum speed, at least 5 touches. Add 10 µL of conjugated nanobeads per 10^7 cells. Immediately, mix well to fully incorporate, and incubate on ice for 15 minutes.;Wash the sample with separation buffer and gently mixing. Centrifuge the cell suspension at 300xg for 5 minutes and discard the supernatant.;Resuspend well in 1 mL of separation buffer by pipetting and place on ice.;Note: We recommend a maximum cell density of 1 x 10^8 million cells/mL of buffer (equivalent to 10 tests/mL of buffer) for column loading. A lower cell density may improve recovery. User may need to optimize cell loading density. If working with larger cell sample volumes, sample may need to be loaded in multiple smaller volumes to avoid overloading the column or on multiple columns and pooled after separation. ;Prepare the column by placing in the appropriate corresponding magnetic separator and rinsing with 3 mL separation buffer. Discard the flow through.;Place an appropriate tube below the column to collect the negative fraction. Add the labeled cell suspension to the column and collect the fraction containing the unlabeled cells.;Optional: Filter cell suspension through a sterile 40 µm filter before addition to column.;Wash the column 3 times with 3 mL of separation buffer and collect the flow-through containing the unlabeled cells. Wait until the previous buffer has drained from the reservoir before proceeding to the next wash but avoid letting the column run dry. The negative fraction flow-through may be useful as controls, to monitor purity/yield, or other purposes.;Remove the column from the magnet and place in a collection tube. Allow the column to demagnetize fro 1-2 minutes. Add 5 mL separation buffer to the column reservoir and flush the magnetically labeled fraction into the collection tube with a plunger or supplied device. This contains the magnetically labeled cells; do not discard.;