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Purpose
This test differentiates immune cell sub-populations via Fluorescence-Activated Cell Sorting.
Description: increased CD4-positive T cell number (MP:0008074), decreased CD4-positive T cell number (MP:0008075), etc., …
This protocol refers to data generated at Harwell up until the IMPC Toronto meeting of October 2013.
Experimental Design
Equipment
· Scissors and forceps for biopsy
· Precision balance
· Calibrated single and multichannel pipettes
· GentleMACS tissue dissociator
· Plate shaker
· Moxi-z Cell counter
· Refrigerated centrifuge
· Flow Cytometer (FACS Canto II)
Supplies
· Microplate 96 U Well PS (SLS #MIC9020)
· Petrie dishes (BD Falcon #353004)
· Dispensing troughs
· Extra long 10µl pipette tips for antibody solutions
· GentleMACS C Tubes (Miltenyi Biotec, #130-096-334)
· 50ml Falcon tubes
· 70μm cell strainers that fit 50ml Falcon tubes (BD Falcon, #352350)
· Moxy-Z Cassettes
· Select Microplate 96 U Well PS (SLS #MIC9020)
· RPMI (SIGMA, #R8758 - 100ml bottles)
· FCS (PAA, #A15-151)
· PBS 10X (SIGMA, #D1408)
· EDTA 0.5M (Invitrogen, #15576-028)
· Collagenase II (Serlabo, #CLS2LS004176), stock solution: 70 mg/mL, aliquoted and stored at –20°c
· DNAse I (SIGMA, #DN25), stock solution: 10 mg/mL, aliquoted and stored at –20°c
· 10x RBC lysis solution (eBiosciences, #00-4300-54)
Procedure
Reagent preparation
· RPMI 2% FCS buffer: for spleen processing steps
· FACS buffer: PBS 1X; EDTA 5 mM; 0.5%FCS (v/v). Stable for up to 1 month in the fridge.
· RBC Lysis buffer: Prepare a 1X solution of 10X eBiosciences solution.
· Stopping buffer: PBS 1X/EDTA 0.1M (37.5g in one litre). Require 300ul per sample.
· Buffer for organ collection: RPMI for organ collection
· Enzyme stock solutions:
DNAse I (10 mg/ml), 10mg in 10ml RPMI/ 2% FSC and freeze into 500 μL aliquots
500 μL Collagenase II (70 mg/ml) 1mg in 14ml RPMI/ 2% FSC and freeze into 500 μL aliquots
· Antibody cocktails for Panels 1 & 2
Protect antibodies and prepared cocktails from direct light.
Each sample will require 50µl of diluted antibody cocktail.
Prepare a minimum volume of 600µl.
Antibody cocktails should be vortexed to ensure homogeneity of the solutions.
MRC Harwell Spleen Flow Cytometry Panels 1 & 2
Panel 1: |
||||||
Marker / Antigen |
Fluorochrome |
Specificity |
Supplier |
Catalogue number |
Size |
Final concentration in cocktail |
Live/dead |
SytoxBlue |
Live / dead |
Invitrogen |
S11348 |
250 ul |
1:10000 |
CD5 |
BV421 |
T-cells, highest on helper T-cells, Not NK (also includes B1 Bcells) |
BD Pharmingen |
562739 |
50 µg |
1/400 |
CD4 |
FITC |
Helper T cells |
BD Pharmingen |
557307 |
0.1 mg |
1:3200 |
CD8 |
PE-CF594 |
Cytotoxic T cells |
Invitrogen/Life Technologies |
MCD0824 |
1 ml |
1:3200 |
CD25 |
APC |
Regulatory T cells |
BD Pharmingen |
557192 |
0.1 mg |
1:800 |
CD62L |
APC-Cy7 |
Level of expression distinguishes naive, effector, and memory T cells |
BD Pharmingen |
560514 |
50 µg |
1:100 |
CD44 |
PE |
Activated CD4+ and CD8+ T cells |
BD Pharmingen |
553134 |
0.1 mg |
1:400 |
CD161 |
PECy7 |
NK cells (as well as NK-T cells) |
BD Pharmingen |
552878 |
0.1 mg |
1:100 |
Panel 2: |
||||||
Marker / Antigen |
Fluorochrome |
Specificity |
Supplier |
Catalogue number |
Size |
Final concentration in cocktail |
Live/dead |
SytoxBlue |
Live / dead |
Invitrogen |
S11348 |
250 ul |
1:10000 |
F4/80 |
PE |
Mature macrophages |
eBiosciences |
12-4801-82 |
100 ug |
1:50 |
CD19 |
BV510 |
Overall B Cells |
BD Horizon |
562956 |
50 µg |
1:800 |
IgD |
APC |
Mature B cells |
BD Pharmingen |
560868 |
50 µg |
1:200 |
Ly6C |
FITC |
Monocytes / Macrophages (also neutrophils and some T-subsets) |
BD Pharmingen |
553104 |
0.5 mg |
1:200 |
Ly6G |
BV421 |
Granulocytes |
BD Pharmingen |
562737 |
50 µg |
1/200 |
CD5 |
BV421 |
T-cells, highest on helper T-cells, Not NK (also includes B1 Bcells) |
BD Pharmingen |
562739 |
50 µg |
1/400 |
CD11b |
PE-CF594 |
Monocytes, dendritic cells |
BD Pharmingen |
562287 |
0.1 mg |
1:800 |
CD11c |
PECy7 |
Dendritic cells (has also been detected on mouse splenic NK cells) |
BD Pharmingen |
558079 |
0.1 mg |
1:100 |
MHCII (anti-Mouse I-A/I-E) |
APC-Cy7 |
Activated Dendritic cells |
Insight Biotechnology |
107628 |
100 ug |
1:400 |
MRC Harwell Gating Strategy Version 1
Mix |
Population |
Subset |
Parameter 1 |
Parameter 2 |
Parameter 3 |
Parameter 4 |
Parameter 5 |
Monitor |
|
|
A |
NK total |
|
CD5- |
CD161+ |
CD44+ |
|
|
% |
Innate |
Gated on live cells |
|
T cells, NKT cells, B1 |
|
CD5+ |
|
|
|
|
% |
|
|
|
|
NKT total |
CD5+ |
CD161+ |
CD44+ |
|
|
% |
Innate like |
|
|
|
NKT Effector |
CD5+ |
CD161+ |
CD44+ |
CD62L- |
|
% |
||
|
|
NKT Resting |
CD5+ |
CD161+ |
CD44+ |
CD62L+ |
|
% |
||
|
|
iNKT |
CD5+ |
CD161+ |
CD44+ |
CD4+ |
|
% |
||
|
|
CD4 T cells total |
CD5+ |
CD4+ |
|
|
|
% |
Helper |
|
|
|
CD4 Effector |
CD5+ |
CD4+ |
CD25- |
CD44+ |
CD62L- |
% |
||
|
|
CD4 Resting/Naive |
CD5+ |
CD4+ |
CD25- |
CD44+ |
CD62L+ |
% |
||
|
|
Tregs |
CD5+ |
CD4+ |
CD25+ |
|
|
% |
Regulatory |
|
|
|
Tregs Effector |
CD5+ |
CD4+ |
CD25+ |
CD44+ |
CD62L- |
% |
||
|
|
Tregs Resting |
CD5+ |
CD4+ |
CD25+ |
CD44+ |
CD62L+ |
% |
||
|
|
CD8 T cells total |
CD5+ |
CD8+ |
|
|
|
% |
Cytotoxic |
|
|
|
CD8 Effector |
CD5+ |
CD8+ |
CD44 high |
CD62L- |
|
% |
||
|
|
CD8 Resting |
CD5+ |
CD8+ |
CD44 high |
CD62L+ |
|
% |
||
|
|
CD8 naive |
CD5+ |
CD8+ |
CD44 low |
CD62L+ |
|
% |
||
|
|
gd + B1 |
CD5+ |
CD4- CD8- |
|
|
|
% |
Innate like |
|
B |
|
|
|
|
|
|
|
|
|
|
|
Neutrophils |
|
CD11b+ |
Ly6G high |
|
|
|
% |
Myeloid |
|
|
Monocytes |
|
CD11b+ |
Ly6G- |
Ly6C high |
|
|
% |
||
|
Macrophage |
|
CD11b+ |
Ly6G- |
Ly6C- |
F4/80+ |
MHCII- low |
% |
||
|
Eosinophils |
|
CD11b+ |
Ly6G- |
Ly6C- |
F4/80- |
SSC High |
% |
||
|
B cell total |
|
CD11b- |
CD19+ |
MHCII+ |
|
|
% |
B cells |
|
|
B2 total |
|
CD11b- |
CD19+ |
MHCII+ |
CD5- |
|
% |
||
|
B2 mature |
Trans. 2+ Trans. 3+ Mature + GC |
CD11b- |
CD19+ |
MHCII+ |
CD5- |
IgD+ |
% |
||
|
B2 immature + MZB |
Trans. 1 + MZB |
CD11b- |
CD19+ |
MHCII+ |
CD5- |
IgD- |
% |
||
|
B1 Total |
|
CD11b- |
CD5+ |
CD19+ |
MHCII+ |
|
% |
||
DCs |
DC total |
|
CD11c+ |
MHCII+ |
|
|
|
% |
DCs |
|
|
pDCs |
|
CD11c+ |
MHCII low |
|
|
|
% |
||
|
cDC CD11b type |
|
CD11c+ |
MHCII+ |
CD11b+ |
|
% |
|||
|
cDC CD8a type |
|
CD11c+ |
MHCII+ |
CD11b- |
|
% |
· Read buffer / dead cell exclusion dye
SytoxBlue at 1:10000 concentration.
Require 200ul per well (i.e. 400ul for each spleen).
· Enzyme cocktail (7X): 500 μL of DNAse I (10 mg/ml), 500 μL Collagenase II (70 mg/ml) into 4 ml RPMI, 2% FCS (v/v). Amount is sufficient for 12 spleens.
Other preparations on the day
· Bring Stop solution and FACS buffer to room temperature.
· Prepare wet ice box.
· Number Falcon tubes, C-Tubes & Eppendorfs for dilutions and set out in racks.
· Place open C-tubes on wet ice and add 2.6ml RPMI with 2%FCS.
Note all centrifuge steps are: 5 min, 290 x g at 4°C
Spleen collection
· Collect the spleen from euthanized mice.
· Remove all fat from the spleen and weight the organ on a petri dish (do not hydrate the organ before weighing it as it would lead to substantial errors in measurement).
· Place the spleen in a 1.5ml eppendorf tube with 1mL of RPMI on ice.
· Once in the lab, transfer each spleen to a GentleMacs C-tube containing 2.6ml RPMI with 2%FCS on ice. Note that if the spleen weight exceeds the recommended value of 250 mg of tissue, transfer only part of the spleen (100 mg).
Spleen dissociation
· Add 400 μL of 7X enzyme cocktail to the GentleMACS C tube already containing 2.6 mL of RPMI/2% FCS and the spleen.
· Clip the tube on GentleMACS dissociator and run the IMPC program located in the Favourites folder (this takes roughly 20 mins).
· Add 300 μL of stopping reaction to block enzymatic digestion and dissociate T/DC interactions.
· Filter through 70um Nylon mesh filter to a 50 mL Falcon tube.
· Wash the GentleMACS C tube with 5ml FACS buffer, filter and pool with flow through from previous step.
· Centrifuge and discard supernatant.
· Resuspend total splenocytes in 1 mL FACS buffer.
Cell counting
· Prepare a 1:300 (3ul in 897) diluted aliquot from 1ml splenocyte suspension gently vortex.
· Perform count using the Moxy-Z cell counter.
· Note down the cell count in a spreadsheet that corrects for dilution and calculates the number of cells per µl.
· Pipette the volume containing exactly 2 million cells to a 96 well plate in horizontal fashion starting from A1 onwards for panel 1 staining.
· Do the same for panel 2 staining in separate wells leaving a few empty rows between the panels to avoid cross contamination.
· Top up to final volume of 100ul using FACS buffer, centrifuge and discard supernatant.
Red blood cell lysis, blocking & staining
· Add the 100ul of lysis buffer to the cell pellet from the previous step.
· Pipette up and down 2-3 times to break up the pellet and ensure complete lysis.
· Incubate on ice for 1 minute.
· Centrifuge, discard supernatant and resuspend in 200ul FACS buffer.
· Again centrifuge and discard S/N and resuspend in 50ul of 1:100 FC block and incubate on ice for 15 mins. Top up to 200ul using FACS buffer after incubation.
· Centrifuge, discard supernatant and resuspend in 200ul FACS buffer.
· In order to eliminate aggregated antibodies from your mix, centrifuge each antibody cocktail for 8 mins at 20,000 g and 4°C.
· Centrifuge, discard supernatant and resuspend in 50ul mAB mix in appropriate wells for individual panels followed by incubation on ice and in the dark for 20 minutes. Top up to 200ul with FACS buffer after incubation.
· Wash twice as in step two and at the end of second cycle resuspend the pellet in 200ul of read buffer (Sytox Blue diluted 1:10000 in FACS buffer).
Analysis
Set up the analyser to acquire 300,000 events in the SSC-W Vs SSC-H singlets gate (P1).