This test differentiates immune cell sub-populations via flow cytometry.
Description | MP Term or Comment |
---|---|
increased CD4-positive T cell number | MP:0008074 |
decreased CD4-positive T cell number | MP:0008075 |
etc.. |
This protocol requires several steps in the collection, preparation and analysis of the samples. Each one is detailed separately below.
Note that two different PBS solutions are required for the protocol below, one with Ca2+ and Mg2+, another without Ca2+ and Mg2+.
Type | Antibody (Marker) | Clone | Fluorochrome |
---|---|---|---|
Required | CD5 | 53-7.3 | BV421 |
Required | CD4 | GK1.5 | FITC |
Required | CD44 | IM7 | PE |
Required | CD8a | 53-6.7 | PE-CF594 |
Required | CD25 | PC61 | APC |
Required | CD161 | PK136 | PE-Cy7 |
Required | CD62L | MEL-14 | APC-Cy7 |
Required | Live/Dead | - | SytoxBlue |
Type | Antibody (Marker) | Clone | Fluorochrome |
---|---|---|---|
Required | CD5 | 53-7.3 | BV421 |
Required | Ly6G | 1A8 | BV421 |
Required | CD19 | 1D3 | BV510 |
Required | Ly6C | AL-21 | FITC |
Required | CD21/CD35 | 7G6 | PE |
Required | CD11b | M1/70 | PerCP-Cy5.5 |
Required | CD11c | HL3 | PE-Cy7 |
Required | CD161 | PK136 | APC |
Required | MHCII | M5/114.15.2 | APC-Cy7 |
Required | Live/Dead | - | SytoxBlue |
Optional | CD23 | B3B4 | BV605 |
Set up the analyser to aim acquire 300,000 viable events (live cells) for each of Panels A and B.
Name | Type | Increment | Option | Unit | Data Type | Derived |
---|---|---|---|---|---|---|
Spleen weight | simpleParameter | g | FLOAT | |||
Live leukocytes (Panel A) - % of total events | simpleParameter | % | FLOAT | |||
T cells (Panel A) - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
NKT cells (panel A) - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
NK cells (Panel A) - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
CD4 T cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
CD8+ T cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
CD4+ NKT cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
CD4- NKT cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Treg cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
CD4+ T helper cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Total events (Panel A) | simpleParameter | INT | ||||
Total events (Panel B) | simpleParameter | INT | ||||
Effector CD4+ T helper cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Resting CD4+ T helper cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Effector CD8+ T cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Naïve CD8+ T cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Resting CD8+ T cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Effector CD4+ NKT cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Resting CD4+ NKT cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Effector CD4- NKT cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Resting CD4- NKT cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Live leukocytes (Panel B) - % of total events (Panel B) | simpleParameter | % | FLOAT | |||
Granulocytes - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
Monocytes - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
Eosinophils - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
NK cells (Panel B) - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
Ly6C+ CD11b- NK cells - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
Ly6C+ CD11b+ NK cells - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
CD11b- NK cells - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
CD11b+ NK cells - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
NKT cells (panel B) - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
Ly6C+ NKT cells - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
T cells (panel B) - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
B cells - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
Conventional DC - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
Effector NK cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Effector Treg cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Resting NK cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
Resting Treg cells - % of live leukocytes (Panel A) | simpleParameter | % | FLOAT | |||
CD4- NKT cells - % of NKT cells (Panel A) | simpleParameter | % | FLOAT | |||
CD4+ NKT cells - % of NKT cells (Panel A) | simpleParameter | % | FLOAT | |||
CD4+ T cells - % of T cells | simpleParameter | % | FLOAT | |||
CD4+ T helper cells - % of CD4 T cells | simpleParameter | % | FLOAT | |||
CD8+ T cells - % of T cells | simpleParameter | % | FLOAT | |||
Effector CD4- NKT cells - % of CD4- NKT cells | simpleParameter | % | FLOAT | |||
Effector CD4+ NKT cells - % of CD4+ NKT cells | simpleParameter | % | FLOAT | |||
Effector CD4+ T helper cells - % of CD4+ T helper cells | simpleParameter | % | FLOAT | |||
Effector CD8+ T cells - % of CD8+ T cells | simpleParameter | % | FLOAT | |||
Effector NK cells - % of NK cells (Panel A) | simpleParameter | % | FLOAT | |||
Effector Treg cells - % of Treg cells | simpleParameter | % | FLOAT | |||
Naïve CD8+ T cells - % of CD8+ T cells | simpleParameter | % | FLOAT | |||
Resting CD4- NKT cells - % of CD4- NKT cells | simpleParameter | % | FLOAT | |||
Resting CD4+ NKT cells - % of CD4+ NKT cells | simpleParameter | % | FLOAT | |||
Resting CD4+ T helper cells - % of CD4+ T helper cells | simpleParameter | % | FLOAT | |||
Resting CD8+ T cells - % of CD8+ T cells | simpleParameter | % | FLOAT | |||
Resting NK cells - % of NK cells (Panel A) | simpleParameter | % | FLOAT | |||
Resting Treg cells - % of Treg cells | simpleParameter | % | FLOAT | |||
Treg cells - % of CD4 T cells | simpleParameter | % | FLOAT | |||
B1a cells - % of B cells (Panel B) | simpleParameter | % | FLOAT | |||
CD11b-high cDC - % of conventional DC (Panel B) | simpleParameter | % | FLOAT | |||
CD11b-low cDC - % of conventional DC (Panel B) | simpleParameter | % | FLOAT | |||
CD161+ B cells - % of live leukocytes (Panel B) | simpleParameter | % | FLOAT | |||
CD11b- NK cells - % of NK cells (Panel B) | simpleParameter | % | FLOAT | |||
CD11b+ NK cells - % of NK cells (Panel B) | simpleParameter | % | FLOAT | |||
CD161+ B cells - % of B cells | simpleParameter | % | FLOAT | |||
Follicular B cells - % of B cells | simpleParameter | % | FLOAT | |||
Ly6C+ CD11b- NK cells - % of NK cells (Panel B) | simpleParameter | % | FLOAT | |||
Ly6C+ CD11b+ NK cells - % of NK cells (Panel B) | simpleParameter | % | FLOAT | |||
Ly6C+ NKT cells - % of NKT cells (Panel B) | simpleParameter | % | FLOAT | |||
Marginal zone B cells - % of B cells | simpleParameter | % | FLOAT | |||
T cells (Panel A) - cell count | simpleParameter | count | INT | |||
CD8+ T cells - cell count | simpleParameter | count | INT | |||
Effector CD8+ T cells - cell count | simpleParameter | count | INT | |||
Resting CD8+ T cells - cell count | simpleParameter | count | INT | |||
Naïve CD8+ T cells - cell count | simpleParameter | count | INT | |||
CD4 T cells - cell count | simpleParameter | count | INT | |||
CD4+ T helper cells - cell count | simpleParameter | count | INT | |||
Effector CD4+ T helper cells - cell count | simpleParameter | count | INT | |||
Resting CD4+ T helper cells - cell count | simpleParameter | count | INT | |||
Treg cells - cell count | simpleParameter | count | INT | |||
Effector Treg cells - cell count | simpleParameter | count | INT | |||
Resting Treg cells - cell count | simpleParameter | count | INT | |||
NKT cells (panel A) - cell count | simpleParameter | count | INT | |||
CD4+ NKT cells - cell count | simpleParameter | count | INT | |||
Effector CD4+ NKT cells - cell count | simpleParameter | count | INT | |||
Resting CD4+ NKT cells - cell count | simpleParameter | count | INT | |||
CD4- NKT cells - cell count | simpleParameter | count | INT | |||
Effector CD4- NKT cells - cell count | simpleParameter | count | INT | |||
Resting CD4- NKT cells - cell count | simpleParameter | count | INT | |||
NK cells (Panel A) - cell count | simpleParameter | count | INT | |||
Effector NK cells - cell count | simpleParameter | count | INT | |||
Resting NK cells - cell count | simpleParameter | count | INT | |||
T cells (panel B) - cell count | simpleParameter | count | INT | |||
NKT cells (panel B) - cell count | simpleParameter | count | INT | |||
Ly6C+ NKT cells - cell count | simpleParameter | count | INT | |||
NK cells (Panel B) - cell count | simpleParameter | count | INT | |||
CD11b- NK cells - cell count | simpleParameter | count | INT | |||
Ly6C+ CD11b- NK cells - cell count | simpleParameter | count | INT | |||
CD11b+ NK cells - cell count | simpleParameter | count | INT | |||
Ly6C+ CD11b+ NK cells - cell count | simpleParameter | count | INT | |||
B cells - cell count | simpleParameter | count | INT | |||
B1a cells - cell count | simpleParameter | count | INT | |||
Follicular B cells - cell count | simpleParameter | count | INT | |||
Marginal zone B cells - cell count | simpleParameter | count | INT | |||
CD161+ B cells - cell count | simpleParameter | count | INT | |||
Conventional DC - cell count | simpleParameter | count | INT | |||
CD11b-low cDC - cell count | simpleParameter | count | INT | |||
CD11b-high cDC - cell count | simpleParameter | count | INT | |||
Monocytes - cell count | simpleParameter | count | INT | |||
Granulocytes - cell count | simpleParameter | count | INT | |||
Eosinophils - cell count | simpleParameter | count | INT | |||
Equipment manufacturer | procedureMetadata | BD Biosciences | TEXT | |||
Equipment model | procedureMetadata | BD LSRFortessa Cell Analyzer | TEXT | |||
CS&T Bead lot | procedureMetadata | TEXT | ||||
Anesthesia | procedureMetadata | Injection narcosis with Medetomidine/Midazolam/Butorphanol | TEXT | |||
Cell digestion | procedureMetadata | manual | TEXT | |||
Cell counting performed | procedureMetadata | pre-lysis | TEXT | |||
Cell counting equipment manufacturer | procedureMetadata | Life Technologies | TEXT | |||
Cell counting equipment model | procedureMetadata | Countess Automated Cell Counter | TEXT | |||
Date and time of sacrifice | procedureMetadata | TEXT | ||||
Date and time of sample preparation | procedureMetadata | TEXT | ||||
Sample storage temperature until analysis (in Celsius) | procedureMetadata | 4 | ℃ | TEXT |