Apr 15, 2025
Intracellular MAP2 staining for flow cytometry
- 1Duke University
- Gersbach Lab
Protocol Citation: Samuel Reisman, Charles Gersbach 2025. Intracellular MAP2 staining for flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l264y3v1y/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: April 10, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 126467
Funders Acknowledgements:
NIH
Grant ID: HG012053
Abstract
This protocols describes methods for intracellular staining of MAP2 for flow cytometry.
Materials
- Warm NDM, trypsin, FBS
- Take eBioscience ICC Fix and 10X perm solution out at RT for at least one hour before using
- Make ~8 mL of 1X perm per tube of cells using molecular bio grade water per sample
○ Also make 10 more mls for block buffer (9mL water + 1mL 10x perm)
4. Block buffer (10ml, make in 1x Perm buffer)
○ 0.2M Glycine (0.1504g)
○ 2.5% FBS (0.25mL)
○ 9.75ml Perm buffer
5. In general, minimize the number of tubes and tips you use, and rinse everything as much as possible to maximize cell yield
| A | B | C | D | |
| NDM (Guo 2014) | Unit | 100 mL | ||
| DMEM/F12 (GIBCO) | mL | 97.5 | ||
| 0.5% FBS (GIBCO) | mL | 0.5 | ||
| 3.5 mM glucose (Sigma)* | mg | 63.056 | ||
| penicillin/streptomycin (GIBCO) | mL | 1 | ||
| N2 supplement (GIBCO) | mL | 1 | ||
| Filter sterilize after combining |
Prepare cells
Prepare cells
Add 100ul FBS to eppendorf tubes to be used for collecting cells
Trypsinize cells (6wp) with 600ul 0.025% trypsin
Incubate 5 minutes
Gently pipette trypsin to dislodge cells
add to FBS
Incubate plate an extra minute at 37C
Rinse cells into tube with 600ul NDM, making sure all cells are recovered. Gently singularize
Spin 1000rpm 5min
Resuspend in 1ml PBS
Spin 1000rpm 5min
Fix, Permeabilize, Stain
Fix, Permeabilize, Stain
Aspirate supernatant
add 50ul fix buffer to cell pellet and mix by pipetting
Incubate in dark at RT for 20m on rocker (cover with foil)
If needing to store: Do 2 PBS rinses, spin down 600xG after each. Store in PBS in dark at 4C
Add 1 ml permeabilization buffer
Pellet cells, spin 1000rpm 5min
Resuspend in 1 ml perm Buffer and count cells
Pellet cells, spin 1000rpm 5min
Resuspend cells in 50 ul block buffer and allow to block 10 min at room temp on rocker
Add 50ul block buffer containing proper amount of antibody
For MAP2 staining: use 1ul a-MAP2-488 antibody per 200,000 cells (ab225316 aka Abcam EPR19691 – 488 Conjugated)
Incubate for 30min at RT on rocker covered in foil. Rock at very slow speed
Add 1ml 1x perm to cell / ab suspension
spin 600g 6 min and aspirate supernatant
Rinse with 1 mL 1x perm
spin 600g for 6 min
Resuspend in 100ul FACS buffer
If there are any doubts about clumping, filter through 40 um filter, and sort cells