Dec 03, 2025
TurboID Primary mBM
This protocol is a draft, published without a DOI.
- Ryan Day1
- 1Washington University in St. Louis
Protocol Citation: Ryan Day 2025. TurboID Primary mBM. protocols.io https://protocols.io/view/turboid-primary-mbm-g5dvby267
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: In development
We are still developing and optimizing this protocol
Created: July 11, 2025
Last Modified: December 03, 2025
Protocol Integer ID: 222357
Keywords: turboid in primary murine bone marrow, primary murine bone marrow, performing turboid, primary mbm protocol
Abstract
Protocol for performing TurboID in primary murine bone marrow
Troubleshooting
Reagents
GP2 293Ts (Takara) (or substitute regular 293Ts, adding gag and pol to transfected plasmid mix)
15 cm tissue culture-treated plates
Retronectin: Takara T100B, 2.5 mg at 1 mg/ml
6 well plates
Lysis buffer: 25mM Tris-HCl, 150mM NaCl, 1% TritonX-100, pH 7.2
Washing Buffer 1: 1% SDS in PBS
Washing Buffer 2: 50mM Na2HPO4, 500mM NaCl, 1% TritonX100, pH 7.4
10 mM biotin: Dissolved in DMSO. 0.024431 grams in 10 ml DMSO
Streptavidin beads: ThermoScientific High Capacity Streptavidin Resin (Catalog 20359)
Sigma protease inhibitor cocktail P8340
DMEM + 10% FCS + P/S
Mouse bone marrow transplant media: RPMI 1640 + 15% FCS + P/S + 100 ng/ml stem cell factor, 10 ng/ml thrombopoietin, 50 ng/ml FLT3 ligand, and 6 ng/ml IL-3
Day 1: Plate 293Ts
Plate GP2 293Ts: plate 14.5e6 cells per 15 cm plate
Day 2: Harvest Mice
Harvest femur, tibia, pelvis, humeri by spinning
ACK lyse
Resuspend BM in mouse BM transplant media and count
Resuspend cells in transplant media at ~2.5 x 10^6 cells/ml and incubate overnight.
Day 2: Transfect 293Ts
Make master mix
| A | B | |
| Reagents | 1X | |
| MIG | 22.5 ug | |
| VSVg | 22.5 ug | |
| Optimem | To 640.3ul | |
| Total | 640.3 ul |
Mix Mirus TransIT LT-1 and OptiMEM in separate tube
- For 15 cm plate, use 67.5 ul Mirus + 300 ul OptiMEM
| A | B | |
| Reagents | 1X | |
| Mirus | 67.5ul | |
| OptiMEM | 300ul |
Mix 640.3 ul plasmid master mix with 367.5 ul Mirus master mix per 293T plate
Incubate at room temperature for 30 minutes
Add ~1 ml of plasmid + Mirus mix to each dish and mix with gentle swirling
Incubate at 37ºC overnight
Day 2: Prepare retronectin plates
Add 1.5 ml of 5 ug/ml retronectin to each well of 6 well plate
- Stock retronectin 1 mg/ml
- 7.5 ul of 1 mg/ml retronectin in 1492.5 ul PBS per well
Swirl to mix and cover bottom surface of well
Incubate at 48 hours at 4ºC
Day 3: Change media on 293T
Change media on transfected 293Ts around 8-10 AM.
Add 18 ml fresh 293T media per plate
Day 3: Lineage Depletion on mice
Count BM cells
Resuspend cell pellet in 80 ul buffer per 2 x 10^7 cells (minimum of 320 ul).
Add 20 ul of Direct Lineage Cell Depletion Cocktail per 2 x 10^7 cells (minimum of 80 ul)
Mix well and incubate for 10 minutes on ice or at 4ºC
Lin deplete using AutoMACS.
Place sample in position A
Negative fraction in position B
Positive fraction in position C
Use program Possel2.
Collect negative fraction
- Maximum column binding capacity = 2e8 labelled cells per column, in Lin- labelling expect most cells to be positive. Divide sample into sets of up to 2e8 to purify on separate columns
- Run rinse between different samples
- If more than 1 column per sample, run a quick rinse between columns from same sample
Resuspend cells in transplant media at 1 x 10^6 cells/ml and incubate overnight.
Day 4: Infection day 1
Verify 293T GFP positivity under microscope
Harvest virus
Filter through 0.45 um filter
Gently add 18 ml 293T media back to 293Ts
Adsorb virus to retronectin-coated plates
Aspirate retronectin from 6 well plates
Wash with 2 ml sterile PBS per well
Add 8-9 ml of virus supernatant to each well
Spin at 2500 x g for 90 minutes at 25°C
Remove virus supernatant into bleach
Infect bone marrow cells day 1
Filter cells over 40 um filter and count
Add 1-2.5 x 10^6 BM cells in 4 ml of murine BM transplant media to each well
Spin at 1000 rpm for 7 minutes in JS-4.750 rotor
Incubate at 37°C overnight
Day 5: Infection day 5
Harvest virus
Filter through 0.45 um filter
Bleach 293Ts
Adsorb virus to retronectin-coated plates
Aspirate retronectin from 6 well plates
Wash with 2 ml sterile PBS
Add 8-9 ml of virus supernatant to each well
Spin at 2500 x g for 90 minutes at 25ºC
Remove virus supernatant into bleach
Infect bone marrow cells
Scrape prior day’s cells using cell scraper, transfer to conical tube
Scrape well again. Wash scraped well with 1 ml murine BM transplant media and add to conical tube
Transfer cells in conical tube to new well
Spin at 1000 rpm for 7 minutes in JS-4.750 rotor
Incubate at 37°C overnight
Day 6: Test transduced cells for GFP
Scrape cells from plate and transfer to new plate
Test aliquot for GFP by flow
Day 7: Sort cells
Count cells
Sort for GFP+ Cells
Plate in transplant media at 1e6/ml based on sorter-reported yields
Day 8: Rest cells
Count cells. Replate at 0.5-1e6/ml
Day 9: Biotinylate and Harvest
Count sorted cells
Harvest small aliquot for GFP flow
Harvest aliquot for Western if desired
Aliquot 1-5e6 cells for biotinylation
Resuspend at 1e6/ml
Add biotin to final concentration of 50 uM
- Stock is 10 mM in DMSO, 1:200 dilution
Incubate for 4 hrs at 37ºC
Harvest cells
Harvest aliquot for Western on more concentrated extract if desired
Pellet cells at 1200 rpm x 5 minutes
Wash x 1 with PBS
Make 1.5 ml lysis buffer + 1X protease cocktail per sample and cool on ice.
Lyse samples in 1000 ul lysis buffer + 1X protease inhibitor cocktail and transfer to Eppendorf tube.
3 X Sonicate lysate at setting 3.5, 8 seconds
- Resting on ice between sonications
Add additional 500 ul lysis buffer
Incubate on ice for 10 minutes.
Spin down lysate 10 minutes at > 13,000 rpm at 4ºC
While lysate is spinning, mix streptavidin agarose resin well by inversion
Aliquot 80 ul beads and 1 ml lysis buffer per sample into tube of sufficient size
Mix well and aliquot into 1 Eppendorf tube per sample
Spin 500 g x 1 minute and aspirate supernatant
After lysate has spun down, aliquot 20 ul for Western and freeze
Transfer remainder of lysate to washed streptavidin beads
- Taking care not to disturb cell residue pellet at bottom of tube
Incubate on rotator at 4ºC overnight
Wash, submit for mass spec
Spin beads 500 g x 1 minute.
Aspirate supernatant
Wash beads with 1 ml washing buffer # 1
Invert 10 times to mix
spin beads at 500 g for 1 minute
Aspirate supernatant
Wash beads with 1 ml of lysis buffer
Invert to mix
spin at 500 g for 1 minute
Aspirate supernatant
Repeat wash with 1 ml lysis buffer
Resuspend in 1 ml of washing buffer 2
Invert to mix
Aliquot 20 ul of solution including beads for Western
spin at 500 g for 1 minute
Aspirate supernatant, leaving thin film of washing buffer at bottom
Submit for mass spec