Dec 01, 2023

Public workspaceFlow cytometry

DOI
dx.doi.org/10.17504/protocols.io.ewov1qm47gr2/v1
  • 1Massachusetts General Hospital, ASAP
Fang Zhou
Open access
DOI: dx.doi.org/10.17504/protocols.io.ewov1qm47gr2/v1
Protocol Citation: Pranay Srivastava 2023. Flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qm47gr2/v1
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: November 30, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 91669
Keywords: ASAPCRN
Abstract
This protocol is to assess immune cell profile inn spleen (Chauhan et al. 2018).
Spleen was removed in a 35 mm petri plate with 5 ml RPMI 1640 and digested mechanically and passed through 70 μm filter screen.
The cell suspension was centrifuged, and the pellet was incubated in RBC lysis buffer. The resulting cell suspension was washed in 1xPBS and blocked with Fc Block (Biolegend, Cat# 101302, 1 μl/50 μl).
The cells were incubated with MC1R (Invitrogen, Cat# PIPA521911, 1.39 µg) antibody followed by fluorophore conjugated primary antibodies for extracellular markers (Biolegend Cat# 101235, CD11b-BV421(0.25 µg); Cat# 127641, Ly6G-BV-650 (0.25 µg); Cat# 128041, Ly6C-BV785 (0.125 µg); Cat# 100516, CD4-APC (0.25 µg); Cat# 100751, CD8a-BV510 (0.5 µg); Cat# 152405, CD19-PerCP-Cy5.5 (0.25 µg); Cat# 102036, CD25-BV605 (0.3 µg)) and AF488 (Invitrogen, Cat# A11034, 1:200).
Zombie dye (Biolegend, Cat# 423101, 1µl/sample) was used to differentiate between live and dead cells. Helper T cells and cytotoxic T cells were identified by CD4+ and CD8+, respectively. CD4+CD25+ cells were used to mark Tregs. CD19+ cells were used as marker for B cells. Monocytes were identified as CD11b+Ly6G-Ly6Chigh cells and neutrophils were marked by CD11b+Ly6C-Ly6G+. For monocytes and neutrophils, CD11b-positive cells were first extracted from the live cell subset by expansion with SSC-A, followed by Ly6C. After expansion with Ly6C and Ly6G, we excluded Ly6G-positive cells. Monocytes were identified as CD11b+Ly6G-Ly6C high cells, and neutrophils were marked by CD11b+Ly6C-Ly6G+.
SORP 5 Laser BD Fortessa X-20 (BD Bioscience) and FlowJo v10.7.1 (Becton Dickson & Company) software was used for data acquisition.