Williams1 project protocol

Inter-strain variation in splenocyte populations for 5 inbred strains (Australia ARC)   (2012)

Williams RB
With: French HJ, Hardy K, Rigby R




Williams1_Protocol

Project protocol — Contents
Workflow and sampling
Equipment and supplies
Reagents and solutions
Procedure for measuring spelenocyte population using flow cytometer
Data
References

Workflow and sampling

Workflow

Step
Procedure performed
Equipment
Data collected
1
 Mice sacrificed by cervical dislocation
dissecting kit
-
2
 Spleen harvested and processed
dissecting kit
-
3
 Splenocytes stained and assessed
flow cytometer
 regulatory T cells, naive T cells, and memory/effector T cells

Equipment and supplies

  • Dissecting kit
  • 0.75µM nylon cell strainers (Becton Dickinson)
  • Flow cytometer, BD Biosciences LSR II (Becton, Dickinson and Company, NJ USA) with BD FACSDiva Software Version 6.1.1 (Becton, Dickinson and Company, NJ USA)
  • FlowJo software 8.8.7 (Treestar, San Carlos, California, USA)
  • 12 x 75 mm polypropylene tubes (USA Scientific 1450-0000FC) or polystyrene tubes (BD Biosciences 352008)
  • Parafilm© all-purpose laboratory film (Alcan Packaging, Neenah, WI)  
  • Eppendorf® tubes 12 x 75 mm sample tubes (USA Scientific, Ocala, FL)

Reagents and solutions

  • 0.9% PBS (phosphate buffered saline solution)
  • Red Blood Cell lysis buffer
  • FACS Buffer (PBS with 2 mM EDTA, 0.02% sodium azide, and 2% FBS)
  • Fc blocking antibody
  • Antibody cocktail (in FACS buffer)
    Fluorochrome Antibody
    Alexa 405 / Pac Blue-A CD44
    PerCP-Cy5-5-A CD8
    PE-A CD25
    FITC-A CD4
    APC-Alexa 750 / APC-Cy7-A B220

Procedure for measuring spelenocyte population using flow cytometer

I. Spleen preparation
a. Mice are sacrificed by cervical dislocation by fully trained personnel.
b. Spleens are removed and suspended immediately in MLC buffer and then placed on ice.
c. Spleens are mashed through 0.75µM nylon cell strainers with a 5 mL syringe barrel.
d. Cell suspensions are washed twice in ice cold phosphate buffered saline (PBS).
e. Supernatant is decanted and the cells are re-suspended in 1 mL of Red Blood Cell lysis buffer and then incubated at room temperature (24°C) for 5 min.
f. Hemolyzed red cells and buffer layer are decanted, PBS+ is added and the remaining splenocytes are washed three more times.
g. Cell suspensions are put on ice for subsequent staining and analysis by flow cytometry.

II. Splenocyte staining for flow cytometry
a. Splenocytes are resuspended in FACS buffer before being aliquoted into 96-well plates to give at least 5x105 cells in each well.
b. Fc blocking antibody (20µL) is added to each well and incubated at 4°C for 20 min.
c. Cells are washed in 160µL FACS buffer and centrifuged for 4 min at 1340 rpm at 4°C.
d. Antibody cocktail is added to each well (30µL) and incubated for 30 min at 4°C in the dark.
e. Cells are washed in 160µL FACS buffer and centrifuged for 4 min at 1340 rpm at 4°C.
f. The cells are then analyzed according to the manufacturers instructions using a BD Biosciences LSR II with BD FACSDiva Software Version 6.1.1.

III. Cell counts acquisition and analysis
a. Samples are acquired on a BD LSR II according to manufacturer’s protocol and acquired cell count files are then analyzed with FlowJo software (see examples).

Submitting investigator's note: These files are available upon request from the investigators.

b. Lymphocytes are characterized based on their forward (FSC-A) and side scatter (SSC-A) signatures, and single cells identified based on their FSC-A and FSC-H signatures.
c. Population gates for CD4+ B220- cells are set and CD4+ CD8- cells are gated from that population (Fig. 1).
d. CD4+ CD8- cells are binned into three populations based on their emission of CD44 and CD25 labeled antibodies. These three groups are CD4+CD25+CD44int CD4+CD25-CD44hi and CD4+CD25-CD44lo (Fig. 1).
e. Data are exported and analyzed in the R statistical package as percent of total lymphocytes and percent of total CD4+ T cells.


Figure 1. Population gates of specific cell population.

Stained cells
FACS staining
Lymphocytes FSC-A, SSC-A
Single cells CD4+, B220-, CD8-
Regulatory T cells CD4+, CD25+, CD44Int
Naive T cells CD4+, CD25-, CD44Lo
Memory/Effector T cells CD4+, CD25-, CD44Hi

Data collected by investigator

CD4 T cells - regulatory (Treg)

percent of lymphocytes

percent of CD4 T cells

CD4 T cells - memory effector

percent of lymphocytes

percent of CD4 T cells

CD4 T cells – naïve

percent of lymphocytes

percent of CD4 T cells