For optimal accuracy, we recommend assessing purity via flow cytometry. The Anti-Human CD138 (Syndecan-1) Antibody, Clone MI15 (Catalog #60003) can be used for this purpose. Note, however, that the antibody clone can be partially blocked. Hence, titration of the antibody will be important when assessing purity of the sample after CD138⁺ plasma cell isolation. The antibody concentration will need to be adjusted until the fluorescent antibody signal reaches a plateau during flow cytometric assessment.

Alternative approaches to to assess purity via flow cytometry are:

1. Intracellular detection of κ (kappa) and λ (lambda) light chains with the appropriate fluorchrome-conjugated anti-human monoclonal antibodies against these respective antigens.
2. Use of fluorochrome-conjugated Anti-Human CD38 Antibody, Clone HIT2 (Catalog #60014) and Anti-Human CD45 Antibody, Clone HI30 (Catalog #60018) to detect CD38⁺CD45^variable cells.
3. Use of a fluorochrome-conjugated secondary antibody, such as Goat Anti-Mouse IgG (H+L) Antibody, Polyclonal (Catalog #60138).

For recommendations on how to include appropriate controls for flow cytometry and the best practices for gating please see the Tech Tip: Considerations for Flow Cytometry Gating

Determining the CD138⁺ plasma cell recovery will provide a more accurate measure of performance of the isolation versus only counting the final yield of the isolated cells. The recovery calculation will take the actual number of CD138⁺ plasma cells in the starting sample into account, while only determining the final yield of the isolated cells will not.

To determine the percentage of CD138⁺ plasma cells that have been recovered from the starting sample after isolation with the kit, please see the Tech Tip: Calculation of Recovery from Cell Isolation Procedures.

The EasySep™ Human CD138 Positive Selection Kit II (Catalog# 17877) has been developed to isolate CD138⁺ plasma cells from mononuclear cells that have been isolated from bone marrow or blood samples via density gradient centrifugation. The EasySep™ Human Whole Blood and Bone Marrow CD138 Positive Selection Kit II (Catalog# 17887) has been developed to isolate CD138⁺ plasma cells directly from bone marrow or blood samples without prior isolation of mononuculear cells.

We do not recommend cryopreservation of bone marrow samples because CD138 protein expression on the surface of plasma cells will be significantly reduced as a result of cryopreservation compared to fresh samples. This will affect the performance of the isolation. Thawing of the sample will also lead to significant lysis of the red blood cells and granulocytes, which will further affect sample quality and downstream isolation of plasma cells. We recommend storing bone marrow samples at room temperature for no longer than 72 hrs instead.

If cryopreservation of bone marrow samples cannot be avoided, one recommendation would be to incubate the thawed cells with DNAse I. CD138⁺ plasma cells can then further be isolated with the EasySep™ Human Whole Blood and Bone Marrow CD138 Positive Selection Kit II. Purity and recovery might not be at par with isolation from fresh samples because of the reasons stated above. For suggestions on how to thaw cryopreserved cells, please see the video protocol, How to Thaw Frozen Human Primary Cells.

Alternatively, mononuclear cells can first be isolated from the thawed bone marrow samples via density gradient centrifugation, followed by isolation of CD138⁺ plasma cells with the EasySep™ Human CD138 Positive Selection Kit II (Catalog# 17877). The density gradient isolation step could potentially reduce the presence of dead/dying cells and debris in the sample post-thaw, which could negatively affect the isolation and purity of CD138⁺ plasma cells.

Yes, RNA and/or DNA can be extracted from the plasma cells after isolation with the kit. To remove the magnetic particles (EasySep™ Dextran RapidSpheres™ 50100) upstream of DNA/RNA extraction, please see the Tech Tip: Acquiring DNA or RNA from Positively Selected Cells.

Yes, the isolated plasma cells can be further analyzed with NGS and flow cytometry. For further details on the compatibility of EasySep™ positive selection kits with downstream applications, please see EasySep™ Performance Data.

No, the magnetic particles cannot be released from the cell surface after plasma cell isolation and will remain on the surface of the isolated cells. However, the presence of magnetic particles on the cell surface of the CD138⁺ plasma cells should not interfere with most common downstream applications, as shown in performance data for other EasySep™ kits.

No sample processing is required when starting with a blood sample. As long as the blood is collected in a tube containing anticoagulant, one can proceed with manual or automated CD138⁺ plasma cell isolation protocol as described in the product information sheet of the kit.

Yes, if desired, an external handheld barcode scanner can be connected to the instrument via one of the USB ports on the front panel. This will allow the user to scan the sample ID barcodes as well as the catalog and lot number of the RoboSep™ Buffer bottle used in the isolation (instead of adding this information manually during the set-up of a run). The catalog and lot numbers of the reagent vials of the kit will be captured automatically via the built-in barcode scanner. Scanning with the built-in scanner will also allow verification that reagents are positioned in the correct slots of the instrument carousel. This built-in scanner can be disabled if needed and a handheld scanner can be used instead. For further information on what handheld scanner to use, please contact techsupport@stemcell.com.