May 28, 2024
Version 2
709.1 Staining of Dissociated Lung Cells for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE sequencing) V.2
- 1Department of Pediatrics, University of Rochester Medical Center;
- 2Genomic Research Center, University of Rochester Medical Center;
- 3University of Rochester;
- 4URMC;
- 5University of Rochester Medical Center
Protocol Citation: Gautam Bandyopadhyay, Jeffrey Malik, Heidie Huyck, Ravi Misra, Gloria S Pryhuber, John Ashton 2024. 709.1 Staining of Dissociated Lung Cells for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE sequencing). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1jk7lmk/v2Version created by Gloria S Pryhuber
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 14, 2024
Last Modified: May 28, 2024
Protocol Integer ID: 100748
Funders Acknowledgement:
NHLBI Molecular Atlas of Lung Development Program Human Tissue Core Grant
Grant ID: U01HL14886
Abstract
This article describes step by step protocol for antibody-staining frozen lung cells for CITE sequencing.
Thawing of frozen dissociated human lung cells
Thawing of frozen dissociated human lung cells
Frozen cells were thawed in 37°C water bath and soon after the freezing medium melted cells were transferred into a 15 ml conical tube and the tubes were filled with staining buffer [1% BSA (w/v, Millipore) in DPBS (Biowhittaker)] to dilute the DMSO in freezing medium.
Cells were centrifuged at 800g, 4°C for 10 minutes.
After centrifugation supernatants were discarded and cells were resuspended in 1.0 ml staining buffer.
Fc receptor blocking and antibody staining
Fc receptor blocking and antibody staining
Viable cell counts were determined by trypan blue exclusion assay
Cells were then incubated in staining buffer containing 2% v/v normal mouse serum (Millipore Sigma) for 10 minutes over ice for Fc-receptor blocking.
Cells were then washed with staining buffer and incubated at a final concentration of 1 × 106 cells per 100 μl of staining cocktail containing analyte-specific antibodies (attached table) for 30 min at 4°C.
Covid Supp CITE Seq Staining Antibody List Gautam B.xlsx14KB
Covid Supp CITE Seq Staining Antibody List Gautam B.xlsx14KB After antibody-staining, cells were washed three times (800g, 4°C for 10 minutes) and resuspended in 100 μl staining buffer and passed through a 100-μm strainer (Falcon, Corning) and sent Genomic Research Center, University of Rochester Medical Center for further processing for CITE sequencing.