FACS HRWL_IMM_001

Jump to:

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).

Parameters & Metadata

Filter:

Name Version Type Description Req. Upload Req. Analysis Annotation Increment Options Ontology Options Unit Data Type Derived History Key Group ID
Name Version Type Description Req. Upload Req. Analysis Annotation Increment Option Ontology Options Unit Data Type Derived History Key Group ID