TotalSeq™ Antibody Cocktail “Spike-in” Guidance

In order to supplement the antibody-targeted proteins assayed in a multiomics experiment, additional liquid single-antibody oligo conjugated TotalSeq™ antibodies, from here on referred to as “spike-in antibodies”, may be added to our lyophilized Totalseq™ cocktails. The recommended procedure is to incorporate the spike-in antibodies into the buffer volume used to reconstitute the antibody cocktail.

 

Warning:

 

TotalSeq™ pre-optimized antibody cocktails are designed for optimal performance at a specific staining volume and cell concentration (designated as a “test size”). Staining performance is assessed only under these specific staining conditions. Alterations to the final staining volume, cell concentration, or targets/analytes may impact panel performance in unpredictable ways. Please also note the recommendation provided below is for use with Totalseq™ catalog cocktails and has not been investigated for custom Totalseq™ antibody cocktails.

 

General Procedure:

 

1. Find the recommended reconstitution volume for your lyophilized TotalSeq™ antibody cocktail in the product Technical Data Sheet (TDS) (27.5 μL of Cell Staining Buffer for most panels).
2. Calculate the amount of each TotalSeq™ antibody needed, per test, for each spike-in antibody based on the desired concentration in the final staining volume. This should be determined in advance by antibody titration. Additional information regarding titration of TotalSeq™ antibodies can be found here: Tips and Tricks for Titrating TotalSeq™ Antibodies.
3. Determine the volume of antibody stock needed for each spike-in TotalSeq™ antibody using the antibody stock concentration (found in the product TDS) and combine together to generate the “spike-in” antibody mixture. Note – Individual TotalSeq™ antibodies are formulated at a concentration of 0.5 μg/μL.
4. Subtract the total volume of the spike-in antibody mixture from the reconstitution volume for the lyophilized TotalSeq™ antibody cocktail in step 1. This is the volume of Cell Staining Buffer to be added to the spike-in antibody mixture. The combination of Cell Staining Buffer and spike-in antibodies is your lyophilized cocktail reconstitution master mix.
5. Follow the protocol for your specific TotalSeq™ lyophilized antibody cocktail in the product TDS, substituting the recommended volume of Cell Staining Buffer with the master mix made in step 4.
6. Following reconstitution, proceed with the recommended cell staining protocol for your respective lyophilized cocktail found in the product TDS.

 

Limitations:

 

1. The amount of spike-in antibody added to reconstitution solution should not exceed 13.75 μg per reaction for TotalSeq™ Universal panels, 25 μg for TotalSeq™ TBNK panels per reaction, and 30 µg per reaction for the TotalSeq™-D Heme Oncology Cocktail.
2. Perform any necessary antibody dilutions in Cell Staining Buffer (Cat. No. 420201). Do not substitute with other buffers or media.
3. Determination of optimal spike-in antibody concentration should be performed prior to spike-in approach. If titration experiments are performed by flow cytometry, additional optimization may be required.

Example Reconstitution Volumes Using 10 μL of Spike-In Antibodies

 

TotalSeq™ Cocktail	Cell Staining Buffer (μL)	Spike-In Volume (μL)	Cell Staining Buffer + spike-in volume for reconstitution (μL)	No. of cells	Volume of reconstituted cocktail to add to FcR blocked cells (μL)	Volume of FcR blocked cells to stain (μL)	Total staining volume (μL)
TotalSeq™- Universal Cocktails	17.5	10	27.5	5x105	25	25	50
TotalSeq™- TBNK Cocktails	40	10	50	1x106	50	50	100
TotalSeq™-D Heme Oncology Cocktail	50	10	60	1x106	50	50	100

 

Note – This table is only meant to provide an example. Cell Staining Buffer and spike-in volume for reconstitution will vary depending on the volume of the spike-in mixture. Spike-in volume + reconstitution volume should equal the recommended reconstitution volume for each cocktail. Refer to the recommended cell staining protocol for your respective lyophilized cocktail found in the product TDS. Please contact technical support here if you have questions.