6.1. Cell Samples

Single or small number of FACS sorted lymphocytes in 96-well plates (i.e., up to ~500 cells/sample)

1. Prior to FACS, prepare 96-well plates with pre-aliquoted 5 µl of PBS and 2 U/µl of RNasin per well. Note: This ensures the cells are suspended in the liquid, preventing them from getting stuck to the tube surface and dehydrating.
2. Immediately after sorting, remove the plate from the motorized stage and seal it to prevent desiccation. Note: The plastic plate can absorb nucleic acids if it gets too dry.
3. Seal the plate with a heat seal, e.g., Optically Clear Heat Seal, Bio-Rad, Cat. 1814030, in combination with Bio-Rad PX1 PCR Plate Sealer, Cat 181-4000, or a seal designed for storage up to -80°C (e.g., Microseal “C” Film, Bio-Rad, Cat. MSC-1001). Note: This prevents the seal from detaching during storage and shipment, which can expose the cell to lower pH and cause cell death.
4. Then, briefly centrifuge the plate for 10-15 seconds to propel any drops on the side walls into the water. Label the plate, place each plate in an individual Ziplock bag, and store the plate at -80°C.
5. Place the plates (which are inside the Ziplock bag) inside a cardboard box or case and secure it with tape. Provide information indicating what samples are in each plate and what wells were used for sorting, and complete the DriverMap single-cell AIR Sample Submission Form.

*To ensure that the RNA in sorted cells is intact (not degraded) after the sorting process and shipment, Cellecta requests a Control Plate with any T or B cells with each batch of samples shipped. We will perform the PCR with the Control Plate before proceeding with your samples. If the Control Plate shows amplification, we will proceed with your samples. This QC metric ensures that we produce high-quality receptor profiling data for your valuable samples. If the Control Plate does not amplify, we will work with you to troubleshoot the sorting process, sorting reagents, plate storage, and shipping conditions.

DirectCell AIR Assay for 500-50,000 purified lymphocytes

1. Collect cells in 10 μL of 1xPBS plus 2 U/ul of RNAsin, aliquoted in 1.5-ml test tubes. Note: Avoid collecting cells in media or PBS with a high concentration of BSA (e.g., more than 0.1%).
2. Spin down the test tubes at low speed (e.g., 500g for 5 min) to collect cells.
3. Put the test tubes in a cardboard box and a Ziplock bag, freeze, store at -80°C, and ship on dry ice. Clearly label each tube with numbers (from 1 to X), and a short name (at most, 6-8 printed alphanumeric characters), and send us the DriverMap AIR Sample Submission Form with the sample names.

*Note: In rare cases, RNA in sorted cells can be degraded. In order to prevent any RNA degradation during sorting, we recommend using an alternative protocol in the next section.

AIR assay for 1,000-100,000 lymphocytes

1. For larger cell samples with more than 1,000 lymphocyte cells, we recommend sorting cells in 1.5-ml test tubes with 0.35 mL of RLT+ buffer (Qiagen Cat. No.1053393)
2. Spin down the test tubes at low speed (e.g., 500g for 5 min) to collect cells.
3. Put the test tubes in a cardboard box and a Ziplock bag, freeze, store at -80°C, and ship on dry ice. Clearly label each tube with numbers (from 1 to X), and a short name (at most, 6-8 printed alphanumeric characters), and send us the DriverMap AIR Sample Submission Form with the sample names.

*The same protocol could be used for shipping lymphocytes purified by magnetic beads or small tissue samples. We will purify RNA and DNA and perform the AIR assay from the provided samples stabilized in RLT buffer.

Shipment Information

Ship Samples To:
Attn: Alex Chenchik
Driver Map AIR Service
Cellecta, Inc.
320 Logue Ave
Mountain View, CA 94043
Tel: 650-938-3910

*Note: Send the shipment tracking number to orders@cellecta.com, along with a copy of the Sample Submission Form and PO number.