Oct 11, 2024

Public workspace Flow Cytometry Protocol for LAMP1 Staining V.2

This protocol is a draft, published without a DOI.
  • 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
  • 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
  • 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
  • 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
  • asap
Ali Ghoochani
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2024. Flow Cytometry Protocol for LAMP1 Staining. protocols.io https://protocols.io/view/flow-cytometry-protocol-for-lamp1-staining-dpct5iwnVersion created by Ali Ghoochani
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: October 10, 2024
Last Modified: October 11, 2024
Protocol Integer ID: 109683
Abstract
This protocol outlines a detailed procedure for flow cytometry-based staining of lysosome-associated membrane protein 1 (LAMP1) in human cells. 

Materials

  • Trypsin
  • 3% Formaldehyde in PBS
  • 90% Ice-cold methanol
  • 3% BSA in PBS
  • LAMP1 antibody (Rabbit anti-human LAMP1, Cell Signaling Technology, Cat. No. 9091S)
  • Alexa Fluor-647 secondary antibody
  • Wash buffer: PBS + 1% BSA + 0.05% Tween-20
  • Flow cytometer
  • Centrifuge
Cell Lifting and Centrifugation
Cell Lifting and Centrifugation
Lift adherent cells by adding trypsin to the culture plate.
Once cells are detached, centrifuge the cells at ~Centrifigation1000 x g, 00:03:00  to pellet them.

3m
Cell Fixation
Cell Fixation
Resuspend the cell pellet inAmount1 mL of 3% formaldehyde.
Incubate the cells at 3Temperature37 °C for Duration00:10:00 to fix cells.
10m
Centrifuge the cells at Centrifigation1000 x g for Duration00:03:00 and discard the supernatant.
3m
Permeabilization
Permeabilization
Resuspend the cell pellet in Amount1 mL 90% ice-cold methanol while vortexing at low speed to prevent clump formation.
After the incubation, centrifuge the cells at Centrifigation1000 x g, 4°C, 00:03:00 and discard the supernatant.

3m
Wash cell pellet with Amount1 mL 1XPBS. Centrifigation1000 x g, 4°C, 00:03:00 and and discard the supernatant.

3m
Centrifigation
Wash
Critical
Blocking
Blocking
Resuspend the cells in Amount1 mL of 3% BSA (in PBS).
Incubate at Temperature37 °C for Duration00:30:00 to block non-specific binding sites.


30m
Centrifuge the cells at Centrifigation1000 x g, 00:03:00  and discard the supernatant.

3m
Primary Antibody Staining
Primary Antibody Staining
Resuspend the cells in 3% BSA in PBS containing a Amount300 µL 1:1000 dilution of Rabbit anti-human LAMP1 antibody (Cat. No. 9091).

Incubate at Temperature37 °C , for Duration01:00:00 to Duration02:00:00 with intermittent low-speed vortexing to keep the cells in suspension.
3h
Centrifuge the cells at Centrifigation1000 x g, 00:03:00  and discard the supernatant.
3m
Resuspend the cells in wash buffer (PBS + 1% BSA + 0.05% Tween-20) and centrifuge at Centrifigation1000 x g, 00:03:00  and discard the supernatant.
3m
Repeat the wash step once more to remove excess primary antibody. Centrifigation1000 x g, 00:03:00  and discard the supernatant.
3m
Secondary Antibody Staining
Secondary Antibody Staining
Resuspend the cells in 3% BSA in PBS containing a 1:1000 dilution of Alexa Fluor-647 (or any fluorescently labeled secondary antibody compatible with the flow cytometer).
Incubate at Temperature37 °C for Duration00:30:00 to Duration01:00:00
1h 30m
Wash the cells twice with wash buffer to remove unbound secondary antibody. Centrifuge the cells at Centrifigation1000 x g, 00:03:00  and discard the supernatant.
3m
Flow Cytometry
Flow Cytometry
Resuspend the cells in an appropriate volume of wash buffer (Usually Amount500 µL  for flow cytometry analysis.
Measure the intensity of LAMP1 expression using a flow cytometer, detecting the Alexa Fluor-647 signal (or equivalent secondary antibody fluorophore).