BD® OMICS-One NK-Cell Protein Panel

 This reagent is provided lyophilized in a pre-titrated format.

1.        Remove the BD® OMICS-One NK-Cell 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. If not, briefly centrifuge to collect the contents at the tube bottom.

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.        Place the reconstituted antibodies on ice until the cells are ready for staining.

        Note: Reconstitute antibody immediately before cell staining. Prolonged incubation of reconstituted antibody may increase
        the non-specific background.

5.        For BD® AbSeq Ab-Oligo drop-in of 60 plex or lower, prepare the BD® AbSeq labeling MasterMix in 1.5-mL LoBind tube on ice.

        Note: For drop-in with more than 60 plex, reach out to technical support for calculation.

        For sequential labeling with Sample Tags or no Sample Tags, prepare BD® AbSeq labeling MasterMix for drop-ins as follows:

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        Component                           1 sample (µL)       1 sample +          2 samples +

                                                                30% overage (µL)    30% overage (µL)

        Per BD® AbSeq Ab-Oligo              2.0                 2.6                 5.2

        Total of BD® AbSeq Ab-Oligo         2.0 × N*            2.6 × N             5.2 × N

        FBS† (catalog number 554656)        70 – (2.0 x N)      91 – (2.6 x N)      182 – (5.2 x N)

        Total                               70                  91                  182            

        For co-labeling with Sample Tags, prepare BD® AbSeq labeling MasterMix for drop-ins as follows:

        ____________________________________________________________________________________________

        Component                           1 sample (µL)       1 sample +          2 samples +

                                                                30% overage (µL)    30% overage (µL)

        Per BD® AbSeq Ab-Oligo              2.0                 2.6                 5.2

        Total of BD® AbSeq Ab-Oligo         2.0 × N*            2.6 × N             5.2 × N

        FBS† (catalog number 554656)        120 – (2.0 × N)     156 – (2.6 × N)     312 – (5.2 × N)

        Total                               120                 156                 312            

        *  N = number of drop-in antibodies. N = 0 if there are no drop-in antibodies.

        †  FBS = BD Pharmingen™ Stain Buffer.

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.        If 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 NK-Cell Protein Panel solution to make a total volume of 175 μL.

        If 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 NK-Cell Protein Panel 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, we recommend blocking nonspecific Fc Receptor–mediated false-positive

        signals with Human BD Fc Block (catalog number 564220).

        a.        To perform blocking, pipet the Fc Block MasterMix into a new 1.5-mL LoBind tube on ice:

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                Component                           1 sample (µL)*    1 sample + 20% overage (µL)

                FBS† (catalog number 554656)        20.0              24.0

                Fc Block‡ (catalog number 564220)   5.0               6.0

                Total                               25.0              30.0                      

                *  Sufficient for up to 1 million cells. To block more cells, adjust the volume.

                †  FBS = BD Pharmingen™ Stain Buffer.

                ‡ Fc Block =  BD Pharmingen™ Human BD Fc Block.

        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 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 8 into the cell suspension. Pipet-mix and proceed to Step 12.

11.        Remove the supernatant from the cells without disturbing the pellet. Add 25 µL Stain Buffer (FBS) to the 175 µL of BD® AbSeq labeling
        MasterMix from Step 8 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 3–4 mL Stain Buffer (FBS) to labelled cells and pipet-mix.

15.        Centrifuge at 400 x g for 5 minutes.

16.        Uncap the 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 three washes.

18.        Resuspend the final washed cell pellet in 620 µL cold Sample Buffer from the BD Rhapsody™ Enhanced Cartridge Reagent V3
        (catalog number 667052) and proceed to single cell capture with on-cartridge washing described in substeps a–c. Refer to the
        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.

        Note: Perform on-cartridge washing after cell settling (8 minute incubation) as follows:

        a.        At the protocol section of "Loading cells in BD Rhapsody™ 8-Lane Cartridge", after cell load, incubate the cartridge in the dark
                at room temperature for 8 minutes.

        b.        Place the cartridge on the BD Rhapsody™ HT Xpress and perform the On-Cartridge Wash steps listed as follows:

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                Material to load        Volume (µL) 1 lane      Pipette Mode

                Air                     380                     Prime/Wash

                Cold Sample Buffer      380                     Prime/Wash

                Air                     380                     Prime/Wash

                Cold Sample Buffer      380                     Prime/Wash  

        c.        (Optional) Perform the scanner step: Cell Load Scan, if using BD Rhapsody™ HT Single-Cell Analysis System Single-Cell
                Capture and cDNA Synthesis Protocol (Doc ID 23-24252). No need for 8-minute delay before scanning.

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.

List of all 30 Human AbSeq specificities included in the BD® OMICS-One T-Cell panel:

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Specificity     Clone      Oligo ID     BD® AbSeq Barcode Sequence          

CD11b           M1/70      AHS0005      ATCGTTATTCGTTGTAGTTCGCCCGGTTTGAGTAGT

CD56            NCAM16.2   AHS0019      AGAGGTTGAGTCGTAATAATAATCGGAAGGCGTTGG

CD38            HIT2       AHS0022      GTCAACGATGGGTAGCGGTAGAAATAACGGAACTGG

CD27            M-T271     AHS0025      TGTCCGGTTTAGCGAATTGGGTTGAGTCACGTAGGT

CD2             RPA-2.10   AHS0029      AAACGTAGATTAGAGCCGGGTATGTCGCAACTGATT

CD16            3G8        AHS0053      TAAATCTAATCGCGGTAACATAACGGTGGGTAAGGT

CD184 (CXCR4)   12G5       AHS0060      CAGTGTTTAGAGCGGGTTGCATATGTCGTTTAGAGG

CD49d           9F10       AHS0063      TAGGGTGACTTAGCGATTGATGCGTATGTTTGGGCG

CD314 (NKG2D)   1D11       AHS0065      TTGAAATGCGATGAGACGTAGAGCGATGTAGGTAGC

CD335 (NKP46)   9E2/NKP46  AHS0068      CAATTTGTTCGCGTTTAGTAGTCGTCGTCTTATGGG

CD54            HA58       AHS0076      AAGAGAATATATGCGTGCGTTGTTAAGGGAATGCGT

CD226           DX11       AHS0079      GAGTTTATGATTCGTTTCTTCGGTAGTTCGTCGCTT

CD94            HP-3D9     AHS0085      GAGGTTAGGATAGGTGTACGGGTCGAGTTGAATTCT

CD336 (NKP44)   p44-8      AHS0090      AATGCAAACGATATCACGAAGGGTAGTACACGACGG

CD49a           SR84       AHS0101      ATGACACGAATGCGACGAGAGGCGAAATAGGTTGGT

CX3CR1          2A9-1      AHS0125      GGGTTCACGAGGTTTAAAGCGGTAGTATAGGATGCC

CD122           Mik-β3     AHS0146      TTAAAGAGATTCGTGGGTATTGGCGCAGTCATTCCT

CD140b (PDGFR)  28D4       AHS0151      GACAACATTTAGGACGTGACGAGAGAGTATAGCTTC

CD248           B1/35      AHS0156      ATCACTTATTTCGTTTGGAGGGTTCGTAGGCGTTGC

CD63            H5C6       AHS0157      TGCAGCGTTAGGACCAAGCGTTTACCGTAGAATATT

CD140a          α-R1       AHS0160      TTACTGACTTTCGGACGTTGGTTACTTAGGGTTATG

CD31 (PECAM1)   WM59       AHS0170      CTAAGGGACGTAATTGAGTTTCGGTGATCGCAGTTT

CD96            6F9        AHS0194      CTAATGTAAGAGCGGACGTTTGGGCACTATATGTTT

CD161 (KLRB1)   HP-3G10    AHS0205      TTTAGGACGATTAGTTGTGCGGCATAGGAGGTGTTC

CD158b (KIR)    DX27       AHS0209      CGTAGGAGGATTTCGTCGATGGGTTTGTTAGCGTTC

CD158e1         DX9        AHS0211      AGGTTCATTGCGGCATTAGGCGTCATATAGTAGGTG

CD337/NKp30     P30-15     AHS0213      GGTAACTGACATGACGGAGCGATAATTTCTGGCGGT

CD3             UCHT1      AHS0231      AGCTAGGTGTTATCGGCAAGTTGTACGGTGAAGTCG

CD329(Siglec-9) E10-286    AHS0239      CGGGCGCGAAGATAGGATAATAGGTAACGTCAAATG

CD106           51-10C9    AHS0251      TCTGATTTAGCGGGTGGACGTATTATAGTGATTGGC

1. This reagent is provided lyophilized in a pre-titrated format.
2. The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
3. Go to https://www.bdbiosciences.com/en-us/resources/protocols/single-cell-multiomics for technical protocols.
4. Go to https://abseq-ref-gen.genomics.bd.com/to access AbSeq reference files in FASTA format for bioinformatics analyses.
5. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing. Follow state and local guidelines when disposing of hazardous waste.
6. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
7. For U.S. patents that may apply, see bd.com/patents.
8. Read and understand the safety data sheets (SDSs) before handling chemicals.To obtain SDSs, go to regdocs.bd.com or contact BD Biosciences technical support at scomix@bd.com.