Description
This reagent is provided lyophilized in a pre-titrated format 1. Remove the BD® OMICS-One Immune Profiler Protein Panel tube from the foil bag and bring up to room temperature for 5 minutes. 2. Make sure the pellet is located at the bottom of the tube. 3. Add 35 µL of nuclease-free water to the bottom of the tube and allow antibodies to reconstitute for 5 minutes at room temperature. 4. Store the reconstituted antibodies on ice until the cells are ready for staining. Note: Reconstituted antibodies should be used within 24 hours from reconstitution of the lyophilized format. 5. For drop-in of 60 plex or lower, prepare the BD® AbSeq labeling MasterMix in a 1.5-mL LoBind tube on ice using the procedure in Table 1 for sequential labeling with or without Sample Tags, or the procedure in Table 2 for co-labeling with Sample Tags. Note: For drop-in with more than 60 plex, reach out to technical support for calculation. 6. Pipet-mix the BD® AbSeq labeling MasterMix for drop-ins. Briefly centrifuge to collect the contents at the bottom, and place back on ice. 7. For sequential labeling with Sample Tags or no Sample Tags, for each sample, add 140 μL BD® AbSeq labeling MasterMix of drop-ins to the tube containing 35 μL reconstituted IP solution to make a total volume of 175 μL. For co-labeling with Sample Tags, for each sample, add 120 μL BD® AbSeq labeling MasterMix of drop-ins and 20 μL Sample Tag to the tube containing 35 μL reconstituted IP solution to make a total volume of 175 μL. 8. Pipet-mix the mixture, briefly centrifuge to collect the contents at the tube bottom, and place back on ice. 9. Centrifuge cells at 400 × g for 5 minutes. If Fc Block is used, proceed to step 10. If Fc Block is not used. skip to step 11. 10. (Optional) For samples containing myeloid and B lymphocytes, BD Biosciences recommends blocking nonspecific Fc Receptor–mediated false-positive signals with Human BD Fc Block (Cat. No. 564220). a. To perform blocking, prepare the Fc Block MasterMix in a new 1.5-mL LoBind tube on ice. See Table 3. b. Pipet-mix the Fc Block MasterMix and briefly centrifuge. Place on ice. c. Remove the supernatant from the cells without disturbing the pellet. d. Resuspend the cells from Step 9 in 25 μL of Fc Block MasterMix. e. Incubate the cells at room temperature (15°C to 25°C) for 10 minutes f. Add 175 μL of BD® AbSeq labeling MasterMix from Step 7 into the cell suspension. Pipet-mix and proceed to Step 12. 11. Remove the supernatant from the cells prepared in step 9 without disturbing the pellet. Add 25 μL Stain Buffer (FBS) to the 175 μL of BD® AbSeq labeling MasterMix from Step 7 to make a total volume of 200 μL. Resuspend the cell pellet in 200 μL total volume. Pipet-mix. 12. Transfer the cells with BD® AbSeq labeling MasterMix into a new 5-mL polystyrene Falcon tube. 13. Stain the cells on ice for 30 minutes. 14. Add 3 mL Stain Buffer (FBS) to labelled cells and pipet-mix. 15. Centrifuge at 400 x g for 5 minutes. 16. Uncap each tube and invert to decant supernatant into biohazardous waste. Keep the tube inverted and gently blot on a lint-free wiper to remove residual supernatant from tube rim. 17. Repeat steps 14-16 twice more for a total of 3 washes. 18. Resuspend the final washed cell pellet in 620 µL cold Sample Buffer from the BD Rhapsody™ Enhanced Cartridge Reagent V3 (Cat. No. 667052) and proceed to single cell capture. See BD Rhapsody™ HT Single-Cell Analysis System Single-Cell Capture and cDNA Synthesis Protocol (Doc ID 23-24252) or BD Rhapsody™ HT Xpress System Single-Cell Capture and cDNA Synthesis Protocol (Doc ID 23-24253) for additional details. See Table 4 for AbSeq Specificities, Clone Names, Sequence IDs, and Barcode Sequences. The Barcode Sequence and Sequence ID are specific to each individual AbSeq antibody. Warning: All biological specimens and materials are considered biohazardous. Handle as if capable of transmitting infection and dispose using proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves.
Store at 2-8°C and protected from prolonged exposure to light. Do not freeze.
