37% Percoll Solution (= 33% final Percoll)

Dillon Corvino

Percoll density solution for enrichment of leukocytes from digested liver tissue. 37% of isotonic Percoll stock, equivalent to 33% final Percoll.

Published

September 18, 2026

Protocol ID: BUF-PERC-37 Version: v2.1 Author: Dillon Corvino Date: September 18, 2026

Purpose

This buffer is used for density-based enrichment of leukocytes from digested liver tissue. During centrifugation, hepatocytes and debris remain in the supernatant, while leukocyte-enriched pellets form at the bottom of the tube.

“37%” and “33%” are the same solution

This buffer is called 37% because it is 37% of isotonic Percoll stock. The identical solution is 33% Percoll when quoted by final Percoll content. Both numbers are correct — they use different denominators.

SIP — Stock Isotonic Percoll, also written Standard Isotonic Percoll — is the conventional term for neat Percoll pre-adjusted to physiological osmolality with 10x PBS at 9:1. That mix is 90% Percoll by volume, so:

\[\text{final Percoll \%} = \text{SIP \%} \times 0.9\]

Convention	This buffer	What the % is measured against
% of isotonic stock (SIP)	37.0%	Volume of SIP as a fraction of the working solution
% final Percoll	33.3%	Volume of neat Percoll as a fraction of the working solution

Important

When comparing against an external protocol, check which convention it uses before changing any number.

This buffer and MUS-LIV-DIG quote % of isotonic stock.
The AG Surace SOP #001 quotes % final Percoll — its “33% Percoll” (9 mL Percoll + 1 mL 10x DPBS + 17 mL 1x DPBS) is the same density as this buffer.

Relabelling “37%” as “33%” without also changing the recipe would drop the gradient to 29.7% final Percoll and increase hepatocyte carryover.

Density vs osmolality (important conceptual note)

Percoll provides density, not physiological salt content.
On its own, Percoll is hypotonic and unsuitable for live cells.

Density determines where cells separate during centrifugation.
Osmolality determines whether cells survive the procedure.

To maintain physiological (1x PBS) osmolality, Percoll must be rendered isotonic by adding concentrated salts (10x PBS).
Once Percoll stock has been adjusted to isotonic conditions, all subsequent working solutions can be prepared by dilution with 1x PBS only.

Linked protocols

MUS-IEL-ISO
MUS-LIV-DIG

Preparation of isotonic Percoll stock (required once per bottle)

Percoll stock must be rendered isotonic before use. This step is performed once per bottle and must be done under sterile conditions.

Preparation from a 1000 mL Percoll bottle

Under a biosafety cabinet, using sterile technique:
- Remove 100 mL Percoll from the original 1000 mL bottle.
- Transfer this 100 mL into a clean, sterile glass bottle (retain for later use).
To the original Percoll bottle, add 100 mL sterile 10x PBS.
Cap securely and mix thoroughly by gentle inversion.
Clearly label the bottle with:
- “Isotonic Percoll”
- “+100 mL 10x PBS”
- Date of preparation
- Name or initials
- “Sterile”

The remaining volume (~1000 mL) is now isotonic Percoll stock.

Making the reserved 100 mL Percoll aliquot isotonic

To the previously removed 100 mL Percoll aliquot, add:
- 11.11 mL sterile 10x PBS
Mix gently by inversion until homogeneous.
Label clearly with:
- “Isotonic Percoll”
- “+11.11 mL 10x PBS”
- Date
- Name or initials
- “Sterile”

This aliquot is now functionally identical to the main isotonic Percoll stock and can be used interchangeably.

Warning

Percoll stock and all derived solutions must be kept sterile.

Important note on isotonic Percoll usage

All working Percoll solutions in this protocol assume that input Percoll is already isotonic.

Therefore: - Do not add 10x PBS when preparing 37% Percoll working solutions. - Only dilution with sterile 1x PBS is required.

Working buffer composition

Component	Stock	Volume for 10 mL	Final composition	Notes
Percoll (isotonic)	100%	3.70 mL	37% of SIP = 33.3% final Percoll	Pre-adjusted stock
PBS 1x	—	6.30 mL	—	Diluent

Preparation of working solution

Equilibrate isotonic Percoll stock to room temperature.
Combine isotonic Percoll and sterile 1x PBS in the ratio 3.70 : 6.30, scaled per the table below.
Mix gently by inversion until homogeneous.
Aliquot immediately into labelled tubes — for MUS-LIV-DIG, 25 mL per 50 mL conical tube, one per liver.

Volumes below are the exact requirement per number of livers — see the tip beneath the table on adding margin.

Livers	Total (mL)	Isotonic Percoll (mL)	PBS 1x (mL)	Final Percoll
1	25	9.25	15.75	33.3%
2	50	18.50	31.50	33.3%
3	75	27.75	47.25	33.3%
4	100	37.00	63.00	33.3%
5	125	46.25	78.75	33.3%
6	150	55.50	94.50	33.3%
8	200	74.00	126.00	33.3%
9	225	83.25	141.75	33.3%
10	250	92.50	157.50	33.3%
12	300	111.00	189.00	33.3%

Per liver this is 9.25 mL isotonic Percoll + 15.75 mL PBS = 25 mL.

Tip

The table gives the exact volume for the number of livers being processed, with no margin built in. When preparing for more than one liver, add some extra so the last tube is not short — tube dead volume, pipetting loss, and the meniscus left in the stripette all add up across samples.

How much extra is your call: one additional liver’s worth (25 mL) is usually sufficient, and is simplest to work out. For example, for 9 livers read off the 10-liver row.

Note

That 10 : 17 ratio is arithmetically 37.04% SIP / 33.33% final rather than exactly 37.00% / 33.30% — a difference of 0.03 percentage points, which is not meaningful at the bench. It is also exactly the AG Surace SOP recipe (9 mL Percoll + 1 mL 10x DPBS + 17 mL 1x DPBS), confirming the two protocols use the same gradient.

Note

Prepare the working solution fresh on the day of use for optimal separation performance and cell viability.

Storage and stability

37% Percoll working solution: prepare fresh on the day of use.
Same-day storage at room temperature is acceptable.
Do not freeze working solutions.
Isotonic Percoll stock should be stored according to manufacturer’s instructions and maintained under sterile conditions.

How isotonic Percoll calculations work (reference)

This section explains why 10x PBS is added to Percoll and how the required volume is calculated.

Definitions

V_T = total final volume
V_P = volume of Percoll (contains no salts)
V_10x = volume of 10x PBS

The remaining volume is 1x PBS.

Salt balance logic

The final solution must contain the same total amount of salt as V_T mL of 1x PBS.

Salt contributions: - Percoll contributes 0x - 1x PBS contributes 1x - 10x PBS contributes 10x

Step 1: Express total salt content

(V_T - V_P - V_10x) + 10 · V_10x = V_T

Step 2: Combine terms

V_T - V_P + 9 · V_10x = V_T

Step 3: Subtract V_T from both sides

- V_P + 9 · V_10x = 0

Step 4: Rearrange

9 · V_10x = V_P

Final rule

V_10x = V_P / 9

In words:
For every 9 mL of Percoll, add 1 mL of 10x PBS to make the solution isotonic.

Reagent details

Component	Supplier	Cat#	Notes
Percoll (100%)	TBD	TBD	Must be rendered isotonic and kept sterile
PBS 1x	TBD	TBD	Sterile; Ca2+/Mg2+-free recommended
PBS 10x	TBD	TBD	Used once for isotonic adjustment

Safety (brief)

Handle Percoll and PBS solutions with standard laboratory PPE.
Dispose of Percoll-containing waste according to chemical and biological safety regulations.
Avoid skin and eye contact; consult manufacturer SDS for details.

Version history

Version	Date	Author	Change summary
v1.0	2025-11-20	Dillon Corvino	Initial buffer definition (33% Percoll)
v1.1	2025-12-29	Dillon Corvino	Updated to 37% Percoll; clarified isotonic prep
v1.2	2025-12-29	Dillon Corvino	Added stock isotonic prep, math explanation, sterility notes
v2.0	2026-08-05	Dillon Corvino	Density unchanged. Added explicit dual-convention section: 37% of isotonic stock = 33% final Percoll, with the AG Surace SOP #001 equivalence. Volumes for `MUS-LIV-DIG` raised to 25 mL per 50 mL tube (was 10 mL per 15 mL tube); scaling table added.
v2.1	2026-09-18	Dillon Corvino	Migrated to shared summary:/helper architecture (buffer/protocol redesign).