Nov 30, 2021
ImmunoFACS
This protocol is a draft, published without a DOI.
- Florian Noack1,
- Silvia Vangelisti1,
- Boyan Bonev1
- 1Helmholtz Pioneer Campus
Protocol Citation: Florian Noack, Silvia Vangelisti, Boyan Bonev 2021. ImmunoFACS. protocols.io https://protocols.io/view/immunofacs-b2a2qage
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 23, 2021
Last Modified: November 30, 2021
Protocol Integer ID: 55354
Abstract
Protocol to isolate distinct cell populations by immunostaining followed by FAC-sorting.
Materials
2% Formaldehyde solution (8ml)
Dilute 1ml of 16% Formaldehyde solution (ThermoFisher, Cat. N.: 28908) with 7ml PBS
2M Glycine solution (200ml)
Mix 30.024g of Ultrapure Glycine (Invitrogen, Cat. N.: 15527013) with 200ml of PBS
10% Saponin Solution (2.5ml)
Mix 0.25g Saponin (Sigma, Cat. N.: 47036-50G-F) with 2.5ml Nuclease-free water.
PBS with 1% BSA (7.5ml)
1% BSA (ThermoFisher, Cat. N.: AM2618)
1:100 RNAsin plus RNase inhibitor (Promega, Cat. N.: N261A)
PBS
(scale the volume accordingly to how many samples you have)
For 7.5ml
- 1.5ml Ultrapure BSA 5%
- 5.925ml PBS
- 75µl RNAsin plus RNase inhibitor
Wash Buffer (15ml)
0.1% Saponin (10% stock solution, freshly prepared)
0.2% BSA Ultrapure BSA 5% (ThermoFisher, Cat. N.: AM2618)
1:100 RNAsin plus RNase inhibitor (Promega, Cat. N.: N261A)
PBS
(scale the volume accordingly to how many samples you have)
For 15ml
- 150µl 10% Saponin
- 600µl Ultrapure BSA 5%
- 14.1ml PBS
- 150µl RNAsin plus RNase inhibitor
Staining Buffer (5ml)(100 µl reaction volume for maximum 1x10^6 cells)
0.1% Saponin (10% stock solution, freshly prepared)
1% BSA Ultrapure BSA 5% (ThermoFisher, Cat. N.: AM2618)
1:25 RNAsin plus RNase inhibitor (Promega, Cat. N.: N261A)
PBS
(scale the volume accordingly to how many samples you have)
For 5ml
- 50µl 10% Saponin
- 1ml Ultrapure BSA 5%
- 3.75ml PBS
- 200µl RNAsin plus RNase inhibitor
DAPI Buffer (5ml)
1:1000 DAPI 5mg/ml (ThermoFisher, Cat. N.: D1306)
0.5% Ultrapure BSA 5% (ThermoFisher, Cat. N.: AM2618)
1:100 RNAsin plus RNase inhibitor (Promega, Cat. N.: N261A)
PBS
(scale the volume accordingly to how many samples you have)
For 5ml
- 5µl DAPI
- 500µl Ultrapure BSA 5%
- 4.445ml PBS
- 50µl RNAsin plus RNase inhibitor
Resuspension Buffer (5ml)
0.5% Ultrapure BSA 5% (ThermoFisher, Cat. N.: AM2618)
1:25 RNAsin plus RNase inhibitor (Promega, Cat. N.: N261A)
PBS
(scale the volume accordingly to how many samples you have)
For 7.5ml
- 750µl Ultrapure BSA 5%
- 6.45ml PBS
- 300µl RNAsin plus RNase inhibitor
Antibodies dilutions optimal for ~1x10^6 cells:
Pax6-A488 (BD, Cat. N.: 561664) 2,5µl in 100µl
Eomes-PE (BD, Cat. N.: 566749) 3µl in 100µl
bTUBIII-A647 (BD, Cat. N.: 560394) 7.5µl in 100µl
Prepare a single cell solution using the Milteny Dissociation Kit. Count cells and resuspend in PBS to a concentration of 1 million cells per milliliter.
Add freshly prepared 2% Formaldehyde (from a new vial) in PBS to a final concentration of 1% and incubate for 10 minutes at room temperature with slow! rotation
Add 2M glycine solution to a final concentration of 0.2M to quench the reaction and incubate 5 minutes at room temperature with slow! rotation
Centrifuge cells for 5 minutes at 500xg at 4C.
Wash with cold PBS with 1% BSA.
Resuspend in Wash Buffer.
Wash for 10 minutes in the cold room, on the rocker.
Split aliquot for negative control – wash again (5 minutes).
Spin at 2500xg for 3 minutes at 4˚C.
Remove supernatant and add 100µl per 1 million cells of Stain Buffer with primary antibodies. Incubate 1 hour on the rocker in the cold room.
Wash cells 2x5 minutes in Wash Buffer and pellet with 2500xg for 3 minutes @ 4˚C.
Resuspend in DAPI Buffer and incubate for 10-15 minutes in the cold room. Wash with Resuspension Buffer.
Resuspend the cells in Resuspension Buffer at a concentration of ~10 million cells per ml*, pass the cell suspension through a 40µm cell strainer and proceed immediately to FACS-sorting (check staining on slides to test the protocol)
* the optimal concentration depends on the used FACS
Sort the cells into ~20µl of cold PBS with 1% BSA. Sorting should not exceed more than 3-4 hours. Wash the sorting tubes with PBS with 1% BSA beforehand to prevent cells from sticking to the sides.
After sorting cells can be used immediately or pelleted (5 minutes at 2500xg at 4˚C) and snap-frozen in liquid nitrogen for storage at -80˚C.