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#314 Cell Biology Running

HEK293 routine subculture, passage 12 → 13

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cell-culture HEK293 passage subculture viability

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This template captures a routine 1:6 subculture passage of adherent HEK293 cells in a T75 flask, transitioning from passage 12 to passage 13.

Goal: maintain HEK293 working stock at <80% confluence and >90% viability, prepare seeded T75 for next passage, and confirm mycoplasma-negative status within the 30-day testing window.

Method overview: Pre-passage check confirms ~80% confluence by 20× phase-contrast microscopy. Media is aspirated, cells are washed in PBS, dissociated with 3 mL of 0.05% Trypsin-EDTA at 37°C for 3 minutes, quenched in 7 mL of complete DMEM + 10% FBS, pelleted at 300 × g for 5 minutes, resuspended in 12 mL of fresh complete media, counted on a hemocytometer with Trypan Blue exclusion, and seeded at 0.5 × 10⁶ cells into a new T75. Cells return to the 37°C / 5% CO₂ incubator.

Expected: Viability ≥90% by Trypan Blue exclusion. Total cells harvested 2.5-3.5 × 10⁶ from a confluent T75. Seeded flask reaches ~80% confluence again in 3-4 days at the 1:6 split.

Last saved: 34 seconds ago

11 fields loaded from Cell Culture baseline (SOP v3.2)

Culture

Cell line

select

HEK293, HeLa, CHO-K1, Jurkat, K562, A549

Vessel

select

T75, T25, T175, 6-well, 10 cm dish

Media

text

DMEM + 10% FBS + 1% P/S

Passage

Passage number

number

13

Confluency at passage

number

80%

Trypsin volume

number

3.0mL

Split ratio

text

1:6

QC

Cells/mL

number

4.08×10⁵

Viability

number

94%

Mycoplasma last test

text

2026-03-21 (neg.)

Seed density

number

0.5×10⁶ /T75

hek293_p13_microscope.jpg

2.4 MB

hemocytometer_count.csv

8 KB

passage_log.xlsx

92 KB

01

Pre-warm media + PBS + Trypsin to 37°C

0:15

02

Phase-contrast check at 20× — confirm ~80% confluence

~80% confluent ✓ photo attached

0:05

03

Aspirate media, wash 1× with 10 mL pre-warmed PBS, aspirate

0:03

04

Add 3 mL 0.05% Trypsin-EDTA, incubate 37°C × 3 min

Tap to release

0:03

05

Quench with 7 mL complete media, triturate

0:02

06

Transfer to 15 mL conical, spin 300 × g × 5 min

0:07

07

Aspirate supernatant, resuspend pellet in 12 mL complete media

0:03

08

Hemocytometer count w/ Trypan Blue (10 µL cells + 10 µL TB)

163 cells / 4 sq → 4.08×10⁵/mL · viability 94%

0:08

09

Seed 0.5×10⁶ cells into new T75, add complete media to 12 mL

1.23 mL of cell suspension

0:04

10

Return to 37°C / 5% CO₂ incubator, label P13 + date

Awaiting placement

0:02

	A	B	C	D	E	F	G
1	Passage	Date	Confluency	Total cells	Viability	Mycoplasma	Notes
2	P10	2026-03-14	85%	2.9×10⁶	96%	neg 2026-02-28	Healthy
3	P11	2026-03-21	78%	2.6×10⁶	93%	—	Routine
4	P12	2026-03-28	82%	3.1×10⁶	95%	—	Routine
5	P13	2026-04-04	80%	2.9×10⁶	94%	neg 2026-03-21	This passage

EXP-2026-0118 HEK293 thaw + initial passage (P0) M. Khan Jan 18

EXP-2026-0321 Mycoplasma PCR test (negative) M. Khan Mar 21

SOP-0322 Cell Culture Standard Protocol v3.2 QA Team Mar 2

LINE-0007 HEK293 working stock origin + characterization Cell Bank Jan 18

MYCO-0044 Mycoplasma test schedule (30-day window) QA Team Feb 1

DMEM (high glucose, GlutaMAX)

10566016 · Lot 2401983

In stock

Fetal Bovine Serum (heat-inactivated)

A5256701 · Lot 2480142

In stock

Penicillin/Streptomycin (10,000 U/mL)

15140122 · Lot 2511028

In stock

0.05% Trypsin-EDTA

25300062 · Lot 2461022

Low

PBS (no Ca²⁺ / Mg²⁺)

10010023 · Lot 2511442

In stock

Trypan Blue (0.4%)

15250061 · Lot 2491834

In stock

HEK293 working stock (cryovials)

LN₂ vapor · Tank 2 · Rack B · Box HEK293-WS

×24

HEK293 master stock (cryovials)

LN₂ vapor · Tank 1 · Rack A · Box HEK293-MS

×10

Media bottle in use (4 °C)

4 °C · Cold room · Shelf 2

×1

Visibility

Khan LabM. Khan

Can write

M. KhanDr. R. Patel

Color

Stroke width

Eraser

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{
    "experiment": {
        "id": 314,
        "custom_id": "CC-HEK293-P13-2026-04",
        "title": "HEK293 routine subculture, passage 12 → 13",
        "template": "Cell Culture",
        "template_version": "v3.2",
        "category": "Cell Biology",
        "status": "Running",
        "started_on": "2026-06-06",
        "tags": [
            "cell-culture",
            "HEK293",
            "passage",
            "subculture",
            "viability"
        ]
    },
    "main_text": "This template captures a routine 1:6 subculture passage of adherent HEK293 cells in a T75 flask, transitioning from passage 12 to passage 13.\nGoal: maintain HEK293 working stock at <80% confluence and >90% viability, prepare seeded T75 for next passage, and confirm mycoplasma-negative status within the 30-day testing window.\nMethod overview: Pre-passage check confirms ~80% confluence by 20× phase-contrast microscopy. Media is aspirated, cells are washed in PBS, dissociated with 3 mL of 0.05% Trypsin-EDTA at 37°C for 3 minutes, quenched in 7 mL of complete DMEM + 10% FBS, pelleted at 300 × g for 5 minutes, resuspended in 12 mL of fresh complete media, counted on a hemocytometer with Trypan Blue exclusion, and seeded at 0.5 × 10⁶ cells into a new T75. Cells return to the 37°C / 5% CO₂ incubator.\nExpected: Viability ≥90% by Trypan Blue exclusion. Total cells harvested 2.5-3.5 × 10⁶ from a confluent T75. Seeded flask reaches ~80% confluence again in 3-4 days at the 1:6 split.",
    "extra_fields": {
        "Culture": {
            "Cell line": {
                "type": "select",
                "value": "HEK293, HeLa, CHO-K1, Jurkat, K562, A549"
            },
            "Vessel": {
                "type": "select",
                "value": "T75, T25, T175, 6-well, 10 cm dish"
            },
            "Media": {
                "type": "text",
                "value": "DMEM + 10% FBS + 1% P/S"
            }
        },
        "Passage": {
            "Passage number": {
                "type": "number",
                "value": "13"
            },
            "Confluency at passage": {
                "type": "number",
                "value": "80 %"
            },
            "Trypsin volume": {
                "type": "number",
                "value": "3.0 mL"
            },
            "Split ratio": {
                "type": "text",
                "value": "1:6"
            }
        },
        "QC": {
            "Cells/mL": {
                "type": "number",
                "value": "4.08 ×10⁵"
            },
            "Viability": {
                "type": "number",
                "value": "94 %"
            },
            "Mycoplasma last test": {
                "type": "text",
                "value": "2026-03-21 (neg.)"
            },
            "Seed density": {
                "type": "number",
                "value": "0.5 ×10⁶ /T75"
            }
        }
    },
    "attached_files": [
        {
            "filename": "hek293_p13_microscope.jpg",
            "type": "png",
            "size": "2.4 MB"
        },
        {
            "filename": "hemocytometer_count.csv",
            "type": "csv",
            "size": "8 KB"
        },
        {
            "filename": "passage_log.xlsx",
            "type": "xlsx",
            "size": "92 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Pre-warm media + PBS + Trypsin to 37°C",
            "duration": "0:15",
            "complete": true
        },
        {
            "n": 2,
            "description": "Phase-contrast check at 20× — confirm ~80% confluence",
            "duration": "0:05",
            "complete": true,
            "note": "~80% confluent ✓ photo attached"
        },
        {
            "n": 3,
            "description": "Aspirate media, wash 1× with 10 mL pre-warmed PBS, aspirate",
            "duration": "0:03",
            "complete": true
        },
        {
            "n": 4,
            "description": "Add 3 mL 0.05% Trypsin-EDTA, incubate 37°C × 3 min",
            "duration": "0:03",
            "complete": true,
            "note": "Tap to release"
        },
        {
            "n": 5,
            "description": "Quench with 7 mL complete media, triturate",
            "duration": "0:02",
            "complete": true
        },
        {
            "n": 6,
            "description": "Transfer to 15 mL conical, spin 300 × g × 5 min",
            "duration": "0:07",
            "complete": true
        },
        {
            "n": 7,
            "description": "Aspirate supernatant, resuspend pellet in 12 mL complete media",
            "duration": "0:03",
            "complete": true
        },
        {
            "n": 8,
            "description": "Hemocytometer count w/ Trypan Blue (10 µL cells + 10 µL TB)",
            "duration": "0:08",
            "complete": true,
            "note": "163 cells / 4 sq → 4.08×10⁵/mL · viability 94%"
        },
        {
            "n": 9,
            "description": "Seed 0.5×10⁶ cells into new T75, add complete media to 12 mL",
            "duration": "0:04",
            "complete": true,
            "note": "1.23 mL of cell suspension"
        },
        {
            "n": 10,
            "description": "Return to 37°C / 5% CO₂ incubator, label P13 + date",
            "duration": "0:02",
            "complete": false,
            "note": "Awaiting placement"
        }
    ],
    "spreadsheet": [
        [
            "Passage",
            "Date",
            "Confluency",
            "Total cells",
            "Viability",
            "Mycoplasma",
            "Notes"
        ],
        [
            "P10",
            "2026-03-14",
            "85%",
            "2.9×10⁶",
            "96%",
            "neg 2026-02-28",
            "Healthy"
        ],
        [
            "P11",
            "2026-03-21",
            "78%",
            "2.6×10⁶",
            "93%",
            "—",
            "Routine"
        ],
        [
            "P12",
            "2026-03-28",
            "82%",
            "3.1×10⁶",
            "95%",
            "—",
            "Routine"
        ],
        [
            "P13",
            "2026-04-04",
            "80%",
            "2.9×10⁶",
            "94%",
            "neg 2026-03-21",
            "This passage"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0118",
                "title": "HEK293 thaw + initial passage (P0)",
                "owner": "M. Khan",
                "date": "Jan 18"
            },
            {
                "ref_id": "EXP-2026-0321",
                "title": "Mycoplasma PCR test (negative)",
                "owner": "M. Khan",
                "date": "Mar 21"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0322",
                "title": "Cell Culture Standard Protocol v3.2",
                "owner": "QA Team",
                "date": "Mar 2"
            },
            {
                "ref_id": "LINE-0007",
                "title": "HEK293 working stock origin + characterization",
                "owner": "Cell Bank",
                "date": "Jan 18"
            },
            {
                "ref_id": "MYCO-0044",
                "title": "Mycoplasma test schedule (30-day window)",
                "owner": "QA Team",
                "date": "Feb 1"
            }
        ]
    },
    "compounds": [
        {
            "name": "DMEM (high glucose, GlutaMAX)",
            "catalog_lot": "10566016 · Lot 2401983",
            "stock_status": "In stock"
        },
        {
            "name": "Fetal Bovine Serum (heat-inactivated)",
            "catalog_lot": "A5256701 · Lot 2480142",
            "stock_status": "In stock"
        },
        {
            "name": "Penicillin/Streptomycin (10,000 U/mL)",
            "catalog_lot": "15140122 · Lot 2511028",
            "stock_status": "In stock"
        },
        {
            "name": "0.05% Trypsin-EDTA",
            "catalog_lot": "25300062 · Lot 2461022",
            "stock_status": "Low"
        },
        {
            "name": "PBS (no Ca²⁺ / Mg²⁺)",
            "catalog_lot": "10010023 · Lot 2511442",
            "stock_status": "In stock"
        },
        {
            "name": "Trypan Blue (0.4%)",
            "catalog_lot": "15250061 · Lot 2491834",
            "stock_status": "In stock"
        }
    ],
    "storage": [
        {
            "name": "HEK293 working stock (cryovials)",
            "location": "LN₂ vapor · Tank 2 · Rack B · Box HEK293-WS",
            "count": "×24"
        },
        {
            "name": "HEK293 master stock (cryovials)",
            "location": "LN₂ vapor · Tank 1 · Rack A · Box HEK293-MS",
            "count": "×10"
        },
        {
            "name": "Media bottle in use (4 °C)",
            "location": "4 °C · Cold room · Shelf 2",
            "count": "×1"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "Khan Lab"
            },
            {
                "type": "Person",
                "name": "M. Khan"
            }
        ],
        "can_write": [
            {
                "type": "Person",
                "name": "M. Khan"
            },
            {
                "type": "Person",
                "name": "Dr. R. Patel"
            }
        ]
    }
}