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#314 Cell Biology Complete

PBMC T cell phenotyping (CD3/CD4/CD8/CD45)

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flow-cytometry PBMC T-cells immunophenotyping CD4-CD8

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This template captures a five-color T cell phenotyping flow cytometry run on peripheral blood mononuclear cells (PBMCs) from healthy-donor blood.

Goal: quantify CD3+, CD3+CD4+, CD3+CD8+ T cell subset frequencies in 12 donor PBMC samples, calculate CD4:CD8 ratios, and confirm population frequencies are within healthy adult reference ranges.

Method overview: PBMCs are isolated from heparinized whole blood by Ficoll density gradient centrifugation, washed in FACS buffer, viability counted with Trypan Blue, blocked with Fc-block, stained with the five-color panel (CD3-BV421, CD4-FITC, CD8-PE, CD45-PerCP, Live/Dead Near-IR APC) for 30 minutes at 4°C in the dark, washed twice, resuspended in FACS buffer, and acquired on a BD FACSAria III with single-color compensation controls. 100,000 events per sample are collected and exported as FCS 3.0 files for FlowJo analysis.

Expected: CD3+ frequency 60-80% of singlets, CD4:CD8 ratio 1.0-2.5 in healthy adults, viable cells >90% by Live/Dead Near-IR exclusion. Compensation matrix verified by single-color controls within ±5% of identity for diagonal channels.

Last saved: 34 seconds ago

12 fields loaded from PBMC Flow baseline (SOP v2.7)

Sample

Sample source

select

PBMC, whole blood, dissociated tumor, cell line

Donor ID

text

HD-2026-04 (cohort)

Cells stained

number

1.0×10⁶ /tube

Viability before stain

number

94%

Panel

Marker 1

text

CD3-BV421 (1:200)

Marker 2

text

CD4-FITC (1:100)

Marker 3

text

CD8-PE (1:200)

Marker 4

text

CD45-PerCP (1:100)

Viability dye

select

Live/Dead Near-IR, Zombie Aqua, 7-AAD, PI

Acquisition & Gating

Cytometer

select

BD FACSAria III, LSRFortessa, Attune NxT

Total events

number

100000/sample

Compensation method

select

single-color auto, manual, AutoSpill

panel_design.pdf

218 KB

compensation_matrix.csv

4 KB

gated_frequencies.xlsx

64 KB

01

Draw whole blood, isolate PBMCs via Ficoll gradient (15 mL Ficoll under 30 mL blood/PBS)

Buffy coat at interface

1:30

02

Wash PBMCs 2× in FACS buffer (PBS + 2% FBS), count by Trypan Blue

~12×10⁶ PBMCs · viability 94%

0:20

03

Aliquot 1×10⁶ cells per stain tube, pellet, resuspend in 100 µL FACS buffer

0:10

04

Block with 5 µL Fc-block, 10 min RT

0:10

05

Add antibody master mix per panel, 30 min 4°C in dark

Panel + L/D NIR

0:30

06

Wash 2× in FACS buffer (300 × g × 5 min), resuspend in 300 µL FACS buffer

0:15

07

Run single-color comp controls + FMO controls on cytometer

Auto comp computed

0:30

08

Acquire stained samples, 100,000 events per tube

FACSAria III · 488 + 405 nm

1:00

09

Export FCS 3.0 files, transfer to FlowJo workspace

0:10

10

Gate populations (singlets → live → CD45+ → CD3+ → CD4+/CD8+), export frequencies

CD4:CD8 = 1.5

0:45

	A	B	C	D	E	F	G
1	Donor	Singlets	Live %	CD3+ %	CD4+ %	CD8+ %	CD4:CD8
2	HD-01	92,847	89.3	64.2	46.1	22.4	2.06
3	HD-02	93,512	91.7	68.4	42.3	28.7	1.47
4	HD-03	94,201	90.2	61.8	38.9	24.6	1.58
5	HD-04	91,328	88.4	70.1	44.2	30.1	1.47
6	HD-05	93,994	92.1	66.7	41.8	27.3	1.53
7	mean	93,176	90.3	66.2	42.7	26.6	1.62
8	sd	1,162	1.6	3.4	2.9	3.1	0.25

EXP-2026-0403 PBMC isolation + cryopreservation (cohort prep) V. Patel Apr 3

EXP-2026-0327 Panel validation: titration + FMOs V. Patel Mar 27

SOP-0507 PBMC Flow Cytometry SOP v2.7 QA Team May 1

PNL-0019 CD3/CD4/CD8/CD45 panel validation document Panel DB Mar 27

WSP-0008 FlowJo workspace v10.8 (cohort gating template) FlowJo Mar 28

CD3-BV421 (clone OKT3, mouse mAb)

317344 · Lot B354210

In stock

CD4-FITC (clone RPA-T4)

300506 · Lot B362194

In stock

CD8-PE (clone RPA-T8)

301008 · Lot B341872

In stock

CD45-PerCP (clone HI30)

304026 · Lot B358147

In stock

Live/Dead Fixable Near-IR

L10119 · Lot 2412048

Low

Ficoll-Paque PLUS density medium

17144002 · Lot 10295174

In stock

Antibody panel aliquots (working)

4 °C · Antibody Fridge · Shelf 2 · Box FLOW-PNL-04

×5

Frozen PBMC backup vials (cohort)

LN₂ vapor · Tank 3 · Rack C · Box HD-2026-04

×12

FACS buffer (in use)

4 °C · Cold room · Shelf 1

×500 mL

Visibility

Patel LabV. Patel

Can write

V. PatelDr. N. Lee

Color

Stroke width

Eraser

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{
    "experiment": {
        "id": 314,
        "custom_id": "FLOW-PBMC-2026-04",
        "title": "PBMC T cell phenotyping (CD3/CD4/CD8/CD45)",
        "template": "Flow Cytometry",
        "template_version": "v3.2",
        "category": "Cell Biology",
        "status": "Complete",
        "started_on": "2026-05-29",
        "tags": [
            "flow-cytometry",
            "PBMC",
            "T-cells",
            "immunophenotyping",
            "CD4-CD8"
        ]
    },
    "main_text": "This template captures a five-color T cell phenotyping flow cytometry run on peripheral blood mononuclear cells (PBMCs) from healthy-donor blood.\nGoal: quantify CD3+, CD3+CD4+, CD3+CD8+ T cell subset frequencies in 12 donor PBMC samples, calculate CD4:CD8 ratios, and confirm population frequencies are within healthy adult reference ranges.\nMethod overview: PBMCs are isolated from heparinized whole blood by Ficoll density gradient centrifugation, washed in FACS buffer, viability counted with Trypan Blue, blocked with Fc-block, stained with the five-color panel (CD3-BV421, CD4-FITC, CD8-PE, CD45-PerCP, Live/Dead Near-IR APC) for 30 minutes at 4°C in the dark, washed twice, resuspended in FACS buffer, and acquired on a BD FACSAria III with single-color compensation controls. 100,000 events per sample are collected and exported as FCS 3.0 files for FlowJo analysis.\nExpected: CD3+ frequency 60-80% of singlets, CD4:CD8 ratio 1.0-2.5 in healthy adults, viable cells >90% by Live/Dead Near-IR exclusion. Compensation matrix verified by single-color controls within ±5% of identity for diagonal channels.",
    "extra_fields": {
        "Sample": {
            "Sample source": {
                "type": "select",
                "value": "PBMC, whole blood, dissociated tumor, cell line"
            },
            "Donor ID": {
                "type": "text",
                "value": "HD-2026-04 (cohort)"
            },
            "Cells stained": {
                "type": "number",
                "value": "1.0 ×10⁶ /tube"
            },
            "Viability before stain": {
                "type": "number",
                "value": "94 %"
            }
        },
        "Panel": {
            "Marker 1": {
                "type": "text",
                "value": "CD3-BV421 (1:200)"
            },
            "Marker 2": {
                "type": "text",
                "value": "CD4-FITC (1:100)"
            },
            "Marker 3": {
                "type": "text",
                "value": "CD8-PE (1:200)"
            },
            "Marker 4": {
                "type": "text",
                "value": "CD45-PerCP (1:100)"
            },
            "Viability dye": {
                "type": "select",
                "value": "Live/Dead Near-IR, Zombie Aqua, 7-AAD, PI"
            }
        },
        "Acquisition & Gating": {
            "Cytometer": {
                "type": "select",
                "value": "BD FACSAria III, LSRFortessa, Attune NxT"
            },
            "Total events": {
                "type": "number",
                "value": "100000 /sample"
            },
            "Compensation method": {
                "type": "select",
                "value": "single-color auto, manual, AutoSpill"
            }
        }
    },
    "attached_files": [
        {
            "filename": "panel_design.pdf",
            "type": "pdf",
            "size": "218 KB"
        },
        {
            "filename": "compensation_matrix.csv",
            "type": "csv",
            "size": "4 KB"
        },
        {
            "filename": "gated_frequencies.xlsx",
            "type": "xlsx",
            "size": "64 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Draw whole blood, isolate PBMCs via Ficoll gradient (15 mL Ficoll under 30 mL blood/PBS)",
            "duration": "1:30",
            "complete": true,
            "note": "Buffy coat at interface"
        },
        {
            "n": 2,
            "description": "Wash PBMCs 2× in FACS buffer (PBS + 2% FBS), count by Trypan Blue",
            "duration": "0:20",
            "complete": true,
            "note": "~12×10⁶ PBMCs · viability 94%"
        },
        {
            "n": 3,
            "description": "Aliquot 1×10⁶ cells per stain tube, pellet, resuspend in 100 µL FACS buffer",
            "duration": "0:10",
            "complete": true
        },
        {
            "n": 4,
            "description": "Block with 5 µL Fc-block, 10 min RT",
            "duration": "0:10",
            "complete": true
        },
        {
            "n": 5,
            "description": "Add antibody master mix per panel, 30 min 4°C in dark",
            "duration": "0:30",
            "complete": true,
            "note": "Panel + L/D NIR"
        },
        {
            "n": 6,
            "description": "Wash 2× in FACS buffer (300 × g × 5 min), resuspend in 300 µL FACS buffer",
            "duration": "0:15",
            "complete": true
        },
        {
            "n": 7,
            "description": "Run single-color comp controls + FMO controls on cytometer",
            "duration": "0:30",
            "complete": true,
            "note": "Auto comp computed"
        },
        {
            "n": 8,
            "description": "Acquire stained samples, 100,000 events per tube",
            "duration": "1:00",
            "complete": true,
            "note": "FACSAria III · 488 + 405 nm"
        },
        {
            "n": 9,
            "description": "Export FCS 3.0 files, transfer to FlowJo workspace",
            "duration": "0:10",
            "complete": true
        },
        {
            "n": 10,
            "description": "Gate populations (singlets → live → CD45+ → CD3+ → CD4+/CD8+), export frequencies",
            "duration": "0:45",
            "complete": true,
            "note": "CD4:CD8 = 1.5"
        }
    ],
    "spreadsheet": [
        [
            "Donor",
            "Singlets",
            "Live %",
            "CD3+ %",
            "CD4+ %",
            "CD8+ %",
            "CD4:CD8"
        ],
        [
            "HD-01",
            "92,847",
            "89.3",
            "64.2",
            "46.1",
            "22.4",
            "2.06"
        ],
        [
            "HD-02",
            "93,512",
            "91.7",
            "68.4",
            "42.3",
            "28.7",
            "1.47"
        ],
        [
            "HD-03",
            "94,201",
            "90.2",
            "61.8",
            "38.9",
            "24.6",
            "1.58"
        ],
        [
            "HD-04",
            "91,328",
            "88.4",
            "70.1",
            "44.2",
            "30.1",
            "1.47"
        ],
        [
            "HD-05",
            "93,994",
            "92.1",
            "66.7",
            "41.8",
            "27.3",
            "1.53"
        ],
        [
            "mean",
            "93,176",
            "90.3",
            "66.2",
            "42.7",
            "26.6",
            "1.62"
        ],
        [
            "sd",
            "1,162",
            "1.6",
            "3.4",
            "2.9",
            "3.1",
            "0.25"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0403",
                "title": "PBMC isolation + cryopreservation (cohort prep)",
                "owner": "V. Patel",
                "date": "Apr 3"
            },
            {
                "ref_id": "EXP-2026-0327",
                "title": "Panel validation: titration + FMOs",
                "owner": "V. Patel",
                "date": "Mar 27"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0507",
                "title": "PBMC Flow Cytometry SOP v2.7",
                "owner": "QA Team",
                "date": "May 1"
            },
            {
                "ref_id": "PNL-0019",
                "title": "CD3/CD4/CD8/CD45 panel validation document",
                "owner": "Panel DB",
                "date": "Mar 27"
            },
            {
                "ref_id": "WSP-0008",
                "title": "FlowJo workspace v10.8 (cohort gating template)",
                "owner": "FlowJo",
                "date": "Mar 28"
            }
        ]
    },
    "compounds": [
        {
            "name": "CD3-BV421 (clone OKT3, mouse mAb)",
            "catalog_lot": "317344 · Lot B354210",
            "stock_status": "In stock"
        },
        {
            "name": "CD4-FITC (clone RPA-T4)",
            "catalog_lot": "300506 · Lot B362194",
            "stock_status": "In stock"
        },
        {
            "name": "CD8-PE (clone RPA-T8)",
            "catalog_lot": "301008 · Lot B341872",
            "stock_status": "In stock"
        },
        {
            "name": "CD45-PerCP (clone HI30)",
            "catalog_lot": "304026 · Lot B358147",
            "stock_status": "In stock"
        },
        {
            "name": "Live/Dead Fixable Near-IR",
            "catalog_lot": "L10119 · Lot 2412048",
            "stock_status": "Low"
        },
        {
            "name": "Ficoll-Paque PLUS density medium",
            "catalog_lot": "17144002 · Lot 10295174",
            "stock_status": "In stock"
        }
    ],
    "storage": [
        {
            "name": "Antibody panel aliquots (working)",
            "location": "4 °C · Antibody Fridge · Shelf 2 · Box FLOW-PNL-04",
            "count": "×5"
        },
        {
            "name": "Frozen PBMC backup vials (cohort)",
            "location": "LN₂ vapor · Tank 3 · Rack C · Box HD-2026-04",
            "count": "×12"
        },
        {
            "name": "FACS buffer (in use)",
            "location": "4 °C · Cold room · Shelf 1",
            "count": "×500 mL"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "Patel Lab"
            },
            {
                "type": "Person",
                "name": "V. Patel"
            }
        ],
        "can_write": [
            {
                "type": "Person",
                "name": "V. Patel"
            },
            {
                "type": "Person",
                "name": "Dr. N. Lee"
            }
        ]
    }
}