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Flow Cytometry

Major companies
BD
IF staining of intracellular cytokines for flow cytometric analysis
immunofluorescence staining of human cells by lysed whole blood method
cell surface staining of human PBMCs and suspension cell lines.

Millipore

Abcam
indirect flow cytometry protocols
PI staining to assess DNA cell cycle
intracellular proteins staining protocol

Santa Cruz

Biolegend

Invitrogen

R&D Systems

Cell Signaling Technology

Abnova

Pierce-antibodies

Beckman Coulter

eBiosceince

Academic Resources
The Janis V. Giorgi Flow Cytometry Laboratory


Cell Lysis
Cell Lysis by ACK lysis buffer

Cell Staining Buffer
  1. ice cold PBS, 10% FBS, 1% sodium azide. (Abcam)


Cell Fixation
  • Cell surface staining
  1. Paraformaldehyde 0.01% to 1% for 10-15minutes only, 100µL per sample.
  2. Acetone or methanol
  3. ice cold -20℃ 70% ethanol in distilled water.
  • Intracellular staining and permeabilization
  1. Formaldehyde followed by detergents
    • fixation in 0.01% formaldehyde for 10-15minute (stabilize proteins)
    • Triton or NP-40 (0.1% to 1% in PBS) partially dissolve the nuclear membrane. 
    • Tween 20, saponin, Digitonin, & Leucoperm - mild membrane solubilizers. 0.5% in PBS. give large enough pore 
  2. Formaldehyde followed by methanol:
    • 0.01% formaldehyde in PBS
    • 1mL ice cold methanol to each sample. mix gently, place at -20℃ for 10 minutes
    • centrifuge wash twice in PBS 1% BSA
  3. methanol followed by detergent:
    • 1mL ice cold methanol each sample.
    • mix gently, place -20℃ for 10min
    • centrifuge, wash twice in PBS 1% BSA
  4. Acetone fixation and permeabilization:
    1. 1mL ice cold acetone to each sample
    2. mix gently place at -20℃ for 10min
    3. centrigue and wash twice in PBS 1% BSA.



Estimation of membrane potential by flow cytometry

flow cytometric measurement of intracellular pH


Reference
Peripheral blood:
Dan G. Duda, et al., A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood, Nature Protocols, 2, 805-810, 2007.  [DOI:10.1038/nprot.2007.111]
*i think this paper contains good protocols and rationale towards isolating cells from peripheral blood for FACS analysis.
crude gravity sedimentation
isolating the cells
FcR blocking
staining
RBC lysis 
cell fixation

The RBC lysis and cell fixation step is behind the antibodies staining as both process could interfere with the antigen-antibody interaction.


murine blood

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