Protocol for inducing epidermis-specific gene deletion and establishing a UVB-irradiated skin inflammation model

Summary

When skin is exposed to ultraviolet B (UVB) radiation, damaged keratinocytes release double-stranded RNA to trigger an inflammatory response. Here, we present a protocol to induce epidermal-specific Zfp750 gene-deficient mice and establish a UVB-induced skin inflammation model. We detail procedures for detecting gene expressions in the epidermis by quantitative reverse-transcription PCR (RT-qPCR) and analyzing changes in skin immune cell subsets after UVB radiation using flow cytometry.

For complete details on the use and execution of this protocol, please refer to Liu et al.¹

Graphical abstract

Highlights

• •Instructions for inducing epidermal-specific gene deletion in K14-CreER^tam mice
• •Steps on establishing a UVB-irradiated skin inflammation model
• •Guidance on epidermal isolation for gene expression analysis
• •Instructions for preparing skin cell suspension for flow cytometry staining

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

When skin is exposed to ultraviolet B (UVB) radiation, damaged keratinocytes release double-stranded RNA to trigger an inflammatory response. Here, we present a protocol to induce epidermal-specific Zfp750 gene-deficient mice and establish a UVB-induced skin inflammation model. We detail procedures for detecting gene expressions in the epidermis by quantitative reverse-transcription PCR (RT-qPCR) and analyzing changes in skin immune cell subsets after UVB radiation using flow cytometry.

Before you begin

Institutional permissions

Institutional permission and approvals for the animal study should be obtained. All procedures in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego (Protocol #S11051).

Preparation of tamoxifen solutions 1 day before the injection

Timing: 2–3 h

• 1.Preheat 12.5 mL sterile corn oil at 37°C.
• 2.Dissolve 0.3 g tamoxifen in 2.5 mL absolute ethanol in a 50 mL conical tube, pipette several times to mix, and place the tube into a 42°C water bath for 10 min.

Note: Tamoxifen does not need to be fully dissolved after this step.

• 3.Add 12.5 mL pre-warmed corn oil to the 2.5 mL tamoxifen-ethanol solution, gently vortex for 20 s.

Note: The final concentration of tamoxifen used for intraperitoneal injection is 20 mg/mL.

• 4.Wrap the conical tube with aluminum foil to protect from light, place the conical tube in 37°C incubator with shaking for 30–60 min until the tamoxifen is completely dissolved.

Note: Extend incubation time at 37°C to 2 h if there are still some solids inside of the conical tube.

• 5.Aliquot 1 mL of tamoxifen solution in 1.5 mL Eppendorf tubes and store at −20°C for up to 6 months.

Preparation on the day of epidermal isolation

Timing: 20 min

• 6.Prepare 75% ethanol in distilled water for disinfecting scissors and forceps.
• 7.Dissolve the powdered Dispase in sterile HBSS to prepare a 3 mg/mL Dispase digestion buffer, pass the Dispase solution through a 0.22 μm filter.

Note: Dispase solution should be prepared freshly for each use. We recommend using 3 mL of 3 mg/mL Dispase solution for each 2 cm × 2 cm area of skin tissue.

Preparation on the day of skin harvest for flow cytometry

Timing: 20 min

• 8.Prepare the FACS buffer before skin harvest. To make FACS buffer, add 15 mL of Fetal Bovine Serum (FBS) and 5 mL of 0.5 M EDTA solution into 480 mL 1× PBS, keep on ice or store at 4°C for one month.
• 9.
  To make a skin digestion buffer for flow cytometry, follow the recipe shown in materials and equipment.

  • a.
    Dissolve 17.5 mg Liberase, 150 mg Collagenase D and 5 mg DNase I in 50 mL DMEM/high glucose medium (final concentration: 0.35 mg/mL Liberase, 3 mg/mL Collagenase D and 0.1 mg/mL DNase I).
  • b.
    Calculate the total volume of skin digestion buffer based on the number of skin samples and prepare it freshly each time. We recommend using a total of 4 mL of skin digestion buffer for each 2 cm × 2 cm section of skin tissue.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
BV510 anti-mouse IgG2a antibody (clone MOPC-173)	BioLegend	Cat# 400268
Anti-mouse CD16/32 blocking antibody (clone 2.4G2) (1:100)	BD Pharmingen	Cat# 553142
PerCP/Cyanine 5.5 anti-mouse Ly6G antibody (clone 1A8) (1:100)	BioLegend	Cat# 127615
PE/Cyanine7 anti-mouse/human CD11b antibody (clone M1/70) (1:100)	BioLegend	Cat# 101216
APC anti-mouse CD11c antibody (clone N418) (1:100)	BioLegend	Cat# 117310
Alexa Fluor 700 anti-mouse MHC II antibody (clone M5/114.15.2) (1:100)	Invitrogen	Cat# 56-5321-82
PE-CF594 anti-mouse F4/80 antibody (clone BM8) (1:100)	BioLegend	Cat# 123145
APC-eFluor 780 anti-mouse CD4 antibody (clone RM4-5) (1:100)	Invitrogen	Cat# 47-0042-80
PE anti-mouse CD19 antibody (clone 6D5) (1:100)	BioLegend	Cat# 115507
BV605 anti-mouse CD8a antibody (clone 53-6.7) (1:100)	BioLegend	Cat# 100743
FITC anti-mouse CD45 antibody (clone 30-F11) (1:100)	BioLegend	Cat# 103107
Chemicals, peptides, and recombinant proteins
Tamoxifen	Sigma-Aldrich	Cat# T5648
Ethanol	Sigma-Aldrich	Cat# E7023
Corn oil	Sigma-Aldrich	Cat# C8267
Dispase	Gibco	Cat# 17105-041
HBSS	Gibco	Cat# 14175-095
PBS	Corning	Cat# 29-031-CV
Nuclease-free water	Invitrogen	Cat# 10977-015
0.5 M EDTA	Promega	Cat# V4231
Liberase	Roche	Cat# 5401020001
Collagenase D	Roche	Cat# 11088882001
DNase I	Sigma-Aldrich	Cat# D5025
3× BD stabilizing fixative buffer	BD Biosciences	Cat# 338036
β-mercaptoethanol	Millipore	Cat# 444203
Isoflurane	VetOne	Cat# 502017
2× Maxima SYBR Green qPCR master mix	Thermo Scientific	Cat# K0253
Red blood cell lysis buffer	Roche	Cat# 11814389001
OneComp eBeads compensation beads	Invitrogen	Cat# 01-1111-42
Viability dye eFluor 506	eBioscience	Cat# 65-0866
Critical commercial assays
RNeasy Plus mini kit	QIAGEN	Cat# 74134
Maxima cDNA synthesis kit	Thermo Fisher Scientific	Cat# K1642
Cyto-Fast Fix/Perm kit	BioLegend	Cat# 426803
Experimental models: Organisms/strains
Mouse: Zfp750fl/flK14-CreERtam, C57BL/6J, 6–8 weeks, male and female	Liu et al.1	Cyagen Biosciences
Mouse: Zfp750fl/wtK14-CreERtam and Zfp750wt/wtK14-CreERtam, C57BL/6J, 6–8 weeks, male and female	Liu et al.1	Cyagen Biosciences
Mouse: K14-CreERtam, C57BL/6J, 6–8 weeks, male and female	The Jackson Laboratory	Cat# 005107
Oligonucleotides
Primer: Zfp750-forward: TGCCAAGTCTGCATTCCA	Liu et al.1	Integrated DNA Technologies
Primer: Zfp750-reverse: AGGCGTGTTCCCAGTCAG	Liu et al.1	Integrated DNA Technologies
Primer: Hprt-forward: CCCAGCGTCGTGATTAGC	Liu et al.1	Integrated DNA Technologies
Primer: Hprt-reverse: CCTTGAGCACACAGAGGG	Liu et al.1	Integrated DNA Technologies
Software and algorithms
FlowJo v.10.8.1	BD Biosciences	http://www.flowjo.com
CFX Maestro software	Bio-Rad	http://www.bio-rad.com
Other
Fetal bovine serum (FBS)	Gibco	Cat# 5670701
DMEM/high glucose medium	Corning	Cat# 10-013-CV
0.22 μm filter	Merck	Cat# GSWP14250
Rotator	Crystal	Cat# TR-02UA
Scalpel	EXE	Cat# 29550
1 mL syringe	Henke Sass Wolf	Cat# 8300014579
Stainless scissors	VWR	Cat# 82027-578
Stainless forceps	Inox	Cat# 11254-20
60 mm petri dish	NEST	Cat# 705001
100 mm petri dish	NEST	Cat# 704001
15 mL conical tube	Falcon	Cat# 352097
50 mL conical tube	Falcon	Cat# 352098
37°C incubator	Forma Scientific	Cat# 20712
70 μm cell strainer	Fisher Scientific	Cat# 22-363-548
Freezer (−20°C)	VWR	Cat# 76580-194
1.5 mL Eppendorf tubes	VWR	Cat# 76332-064
Aluminum foil	Avantor by VWR	Cat# 89107-726
26-gauge needle	BD Biosciences	Cat# 305110
70% alcohol swab	BD Biosciences	Cat# 326895
Electric shaver	Oneisall	Cat# 26225202-003DE
UVB lamp	Spectroline	Cat# EB-280C
UVB meter	Solarmeter	Cat# Model 6.0
Task wipers	Kimtech	Cat# S-8115
CFX96 real-time system	Bio-Rad	N/A
NanoDrop spectrophotometer	Thermo Scientific	Cat# ND2000
96-well PCR plate	Bio-Rad	Cat# HSP9601
Sealing film	Bio-Rad	Cat# MSB1001
5 mL round-bottom flow tubes	Falcon	Cat# 352235

Materials and equipment

Tamoxifen solution

Reagent	Final concentration	Amount
Tamoxifen	20 mg/mL	0.3 g
Corn oil	N/A	12.5 mL
Absolute ethanol	N/A	2.5 mL
Total	N/A	15 mL

Store at −20°C for up to 6 months.

FACS buffer

Reagent	Final concentration	Amount
FBS	3%	15 mL
0.5 M EDTA	5 mM	5 mL
PBS	N/A	480 mL
Total	N/A	500 mL

Store at 4°C for up to 1 month.

Skin digestion buffer

Reagent	Final concentration	Amount
Liberase	0.35 mg/mL	17.5 mg
Collagenase D	3 mg/mL	150 mg
DNase I	0.1 mg/mL	5 mg
DMEM/high glucose	N/A	50 mL
Total	N/A	50 mL

Store at 4°C or on ice for up to 24 h.

Dispase solution

Reagent	Final concentration	Amount
Dispase	3 mg/mL	150 mg
HBSS	N/A	50 mL
Total	N/A	50 mL

Store at 4°C or on ice for up to 24 h.

1× BD stabilizing fixative buffer

Reagent	Final concentration	Amount
3× BD stabilizing fixative buffer	1×	3 mL
PBS	N/A	6 mL
Total	N/A	9 mL

Store at 4°C or on ice for up to 24 h.

RLT Plus lysis buffer

Reagent	Final concentration	Amount
Buffer RLT Plus	N/A	9.9 mL
β-mercaptoethanol	0.143 M	100 μL
Total	N/A	10 mL

Store at 4°C or on ice for up to 24 h.

Step-by-step method details

Tamoxifen injections to induce epidermal-specific Zfp750 gene deletion

Timing: 14 days

This first part of the protocol describes the treatment of Zfp750^fl/fl K14-CreER^tam mice with tamoxifen to induce Zfp750 gene deletion in the epidermis (Zfp750^−/−). Inject adult male and female Zfp750^fl/fl K14-CreER^tam mice and control (Zfp750^fl/wt K14-CreER^tam or Zfp750^wt/wt K14-CreER^tam) mice aged 6 weeks intraperitoneally (i.p.) with 100 μL of 20 mg/mL tamoxifen suspension for 5 consecutive days. Zfp750^fl/fl K14-CreER^tam mice are generated from crossing Zfp750 ^fl/fl and mated with K14 CreER^tam mice.

• 1.Before injection, thaw tamoxifen aliquots and warm it at 37°C for 10 min.
• 2.
  Load the 1 mL syringe with 150 μL of tamoxifen solution before placing the needle on it.

  • a.
    Invert and flick the syringe, push plunger up slowly to remove bubbles.
  • b.
    Put the 26-gauge needle on the syringe and push plunger to 100 μL scale line.
• 3.Restrain mice appropriately in the head-down position, disinfect the injection site using a 70% alcohol swab.

Note: Use a new needle and syringe for each mouse

• 4.Intraperitoneally inject 100 μL of 20 mg/mL tamoxifen solution into the lower left or lower right quadrant of the abdomen of each mouse for 5 consecutive days.

Note: Inject all control (Zfp750^fl/wt K14-CreER^tam or Zfp750^wt/wt K14-CreER^tam) mice and Zfp750^fl/fl K14-CreER^tam mice with an equal amount of tamoxifen.

CRITICAL: After inserting the needle into the animal, pull back on the plunger to make sure there is negative pressure prior to injecting.

• 5.Put the mice back into its cage and monitor their health condition over the entire time span of the experiment.

Hair depilation on mice

Timing: 20 min/mouse

This section describes how to depilate the dorsal skin of the tamoxifen injected mice that are in the second telogen phase of the hair cycle and will generate skin ready for ultraviolet B (UVB)-induced skin inflammation model.

• 6.9 days post the last tamoxifen injection, anesthetize the mice using isoflurane inhalation in an animal induction chamber.
• 7.Shave hair from the mouse dorsal skin using an electric shaver (Figure 1A).

Note: Do not shave hair from head and limbs, avoid damaging the skin barrier.

• 8.Apply Nair hair remover lotion (Nair) on the shaved dorsal skin and gently rub the skin with fingers for less than 1 min to ensure the cream is evenly distributed on the skin surface (Figure 1B).

Note: Do not leave the hair remover lotion on the mouse skin for more than 5 min as it may lead to additional skin irritation and inflammation which could influence the experimental results.

• 9.Place a soft paper towel on the skin, gently press it and slowly peel it off. Most of the hair will adhere to the paper towel (Figure 1C).
• 10.Remove the remaining hair and extra Nair lotion by gently wiping the skin with a wet paper towel (Figure 1D).

Note: Make sure the skin surface is thoroughly cleaned to avoid any Nair lotion causing additional irritation to the skin. If hair remains on the skin, reapply Nair lotion and rub it for less than 1 min, and then gently remove it using a wet tissue.

CRITICAL: Do not use mice in the anagen phase of hair cycle, as this will affect the experimental results. If the skin appears gray or black after hair removal, it indicates that the hair is in the anagen phase. If the skin appears pink or flesh-colored, it indicates the telogen phase.² Therefore, it is recommended to use adult mice at 7–11 week old so that the hair cycle is in telogen during the duration of the experiment.

Figure 1.

Hair depilation on mice dorsal skin

(A) Shave the dorsal skin of anesthetized mice by using an electronic hair clipper.

(B) Apply Nair lotion on the shaved skin and gently rub it with fingers for less than 1 min.

(C) Remove the hair with a soft paper towel.

(D) Gently wipe the skin with a wet paper towel to remove the remaining hair and Nair lotion.

Calibration and setup of UVB lamp device

Timing: 20 min

This is the step-by-step procedure to set up the UVB device and read the intensity of UVB light using a UVB meter.

• 11.Stack several unused empty tip boxes together and place the UVB lamp (Figure 2A) horizontally on top of the tip boxes (Figure 2B).
• 12.Stack the remaining empty tip boxes and place them directly underneath the UVB lamp. Ensure the height of the stacked tip boxes (h1) is approximately the same as the height of the UVB meter (h2) when placed vertically on the table (Figure 2B).

Note: The intensity of UVB light decreases with increasing distance, therefore, the distance from lamp to UVB meter should be identical to the distance from lamp to mice.

• 13.Turn on the UVB lamp and wait for 10 min until the intensity of UVB light is stabilized.

Note: Wear personal protective equipment while operating experiments under UV light to prevent harm to the skin from UV irradiation.

Figure 2.

Expose mice to UVB radiation

(A) UV lamp used in this protocol.

(B) Set up the UV device and make sure the height of stacked tip boxes (h1) is identical to the height of UVB meter (h2).

(C) Place the UVB meter directly under the lamp to measure the irradiance (mW/cm²).

(D) Place mouse directly under the UV lamp and expose to 200 mJ/cm² UVB dosage for two consecutive days.

(E) Formula used for calculating the exposure time (seconds) of UVB irradiation.

Calculate UVB exposure time

Timing: 5 min

This part of the protocol describes how to calculate UVB exposure time for each mouse.

• 14.After positioning the UV device and calibrating the UVB lamp, place the UVB meter directly underneath the UVB lamp, press the switch button to measure the UVB irradiance (Figure 2C).

Note: Hold the UVB meter steady until the reading number is stable, then record the UVB irradiance.

• 15.
  The formula shown in Figure 2E is used for calculating the exposure time (seconds) of UVB radiation.

  • a.
    Exposure time (seconds) refers to the amount of time a mouse needs to be exposed to UVB light to receive a specific dosage of UVB.
  • b.
    UV dosage (mJ/cm²) represents the total energy delivered by UVB light per unit area.
  • c.
    UVB irradiance (mW/cm²) is the intensity of the UVB light measured in step 14.

Note: In this protocol, we treat depilated mice with 200 mJ/cm² UVB dosage. If the UVB irradiance is 2 mW/cm², the exposure time will be 100 s.

Expose depilated mice to UVB radiation

Timing: 2 days

This is the step-by-step protocol to treat depilated mice with 200 mJ/cm² UVB dosage for two consecutive days.

• 16.Place the depilated isoflurane anesthetized mouse (using a nose cone) under the UVB lamp, cover eyes with ocular lubricant, and expose to UVB for the period of time calculated in step 15 (Figure 2D).
• 17.Put mice back into their cages after the first dose of UVB irradiation.
• 18.On the second day, after installing and calibrating the UV device, use the UVB meter to measure the intensity of UVB, and calculate the UVB exposure time again.
• 19.Anesthetize mice using isoflurane inhalation and place each mouse under the UVB lamp with the exposure duration corresponding to the calculated time (Figure 2D).

Note: Irradiate one mouse at a time with UVB; do not place multiple mice under the device simultaneously. Cover the eyes of mice to prevent ultraviolet light from causing damage to its vision.

CRITICAL: During the UVB irradiation, mice must remain in a stationary state with isoflurane inhalation.

• 20.Put UVB irradiated mice back into its cages.

Epidermal isolation for gene expression analysis

Timing: 1.5 days

This step describes the isolation of epidermis from mouse dorsal skin using Dispase. The isolated epidermis can be used for subsequent RNA extraction, cDNA synthesis, and Real time-qPCR to assess Zfp750 gene knockout efficiency.

• 21.24 h after the last UVB treatment, sacrifice tamoxifen injected Zfp750^fl/fl K14-CreER^tam mice and control mice with carbon dioxide inhalation, followed by cervical dislocation to ensure animals do not perceive pain during this process.
• 22.
  Collect the skin and remove subcutaneous fat layer.

  • a.
    Take a 2 cm × 2 cm area of dorsal skin tissue and rinse it with 75% ethanol for 30 s (Figure 3A).
  • b.
    Wash it with 10 mL of PBS and place it in a new 100-mm Petri dish with the dermis side facing up.
  • c.
    Trim the tissue with sterile scalpel and forceps to remove the subcutaneous fat layer in the 100-mm Petri dish (Figures 3B and 3C).

CRITICAL: Complete removal of the fat is essential for effectively separating the epidermis from the dermis. The yellowish fat layer is easily distinguished by sight.

• 23.Add 3 mL of 3 mg/mL Dispase solution in a 60-mm Petri dish and float skin, epidermis-side up (Figure 3D).
• 24.Incubate overnight at 4°C for 16–18 h.

Note: Incubation time should be less than 22 h.

• 25.Use delicate task wipers to absorb moisture from the edges of the skin before transferring it onto the 60-mm Petri dish (Figure 3E).
• 26.Transfer the skin onto a new 60-mm Petri dish with epidermis-side down and gently peel off the epidermis from the dermis with forceps (Figure 3F).

Note: If Dispase digestion is sufficient and effective, the epidermis side will adhere to the bottom of the dish and can be easily peeled off. If it is hard to separate the epidermis, please refer to troubleshooting problem 1.

Figure 3.

Epidermal isolation for RT-qPCR

(A) Cut a 2 cm × 2 cm area of dorsal skin for epidermal isolation.

(B) Remove the subcutaneous fat layer with scalpel and forceps. The yellowish fat layer has been removed from the upper portion of the skin tissue, while the lower portion still retains the fat layer.

(C) Completely remove the fat layer.

(D) Float the skin tissue with epidermis side facing up in a 3 mg/mL Dispase solution and incubate at 4°C for 16–18 h.

(E) Absorb moisture from the edges of the skin tissue.

(F) Place the skin onto a new 60-mm Petri dish with the epidermis side down and carefully separate the epidermis from the dermis.

(G) Cut the epidermis into a paste-like texture.

Epidermal RNA extraction and cDNA synthesis

Timing: ∼1 h

This step outlines the process of RNA extraction from the isolated epidermis using the RNeasy Plus mini kit and the subsequent synthesis of first-strand cDNA from the purified RNA using the Maxima cDNA synthesis kit.

• 27.Cut the epidermis into small pieces using a scalpel (Figure 3G) and add 600 μL of RLT Plus lysis buffer with β-mercaptoethanol.

Note: Cut the epidermis into the smallest pieces possible so that it is fully dissociated. We recommend cutting it into paste-like textures.

• 28.Transfer the fully dissociated epidermal lysate into a 1.5 mL Eppendorf tube. Thoroughly mix it by pipetting.
• 29.Centrifuge the lysate at 12,000 × g for 5 min and transfer the supernatant to a gDNA Eliminator spin column placed in a 2 mL collection tube.

Note: Do not touch the insoluble pellet when transferring the supernatant to a gDNA Eliminator spin column.

CRITICAL: Centrifugation is a crucial step because it helps remove insoluble pellet and small tissue fragments, preventing them from clogging the column.

• 30.Centrifuge at 12,000 × g for 30 s. Discard the column and keep the flow-through.
• 31.Add 600 μL of 70% ethanol to the flow-through and mix well by pipetting.
• 32.Transfer 700 μL of the sample to an RNeasy spin column placed in a 2 mL collection tube. Centrifuge at 12,000 × g for 15 s and discard the flow-through.
• 33.Transfer the remaining 500 μL of the sample to the same RNeasy spin column. Centrifuge at 12,000 × g for 15 s and discard the flow-through.
• 34.Add 700 μL of Buffer RW1 to the RNeasy spin column. Centrifuge at 12,000 × g for 15 s and discard the flow-through.
• 35.Add 500 μL of Buffer RPE to the RNeasy spin column. Centrifuge at 12,000 × g for 15 s and discard the flow-through.
• 36.Add 500 μL of Buffer RPE to the RNeasy spin column. Centrifuge at 12,000 × g for 2 min and discard the flow-through.
• 37.Place the RNeasy spin column in a new 1.5 mL Eppendorf tube. Add 50 μL RNase-free water directly to the column. Centrifuge at 12,000 × g for 1 min to elute the RNA.
• 38.Use NanoDrop spectrophotometer to measure the concentration of RNA and place RNA samples on ice for cDNA synthesis.

Pause point: RNA samples can be stored at −80°C for months.

• 39.Prepare the cDNA synthesis reaction on ice using volumes as listed below.

cDNA synthesis reaction components

Reagent	Amount
5× Reaction Mix	4 μL
Maxima Enzyme Mix	2 μL
Template RNA, 1 μg	variable (between 1 μL and 14 μL)
RNase-free water	to 20 μL
Total	20 μL

Prepare fresh or store at 4°C for less than 1 day.

• 40.Incubate for 10 min at 25°C followed by 15 min at 50°C to synthesize the cDNAs.
• 41.Stop the reaction by heating at 85°C for 5 min and directly perform RT-qPCR.

Pause point: cDNAs can be stored at −20°C for one year.

RT-qPCR

Timing: 3 h

Analysis of the mRNA expression of Zfp750 in the epidermis of Zfp750^fl/fl K14-CreER^tam and control mice by RT-qPCR. Perform RT-qPCRs using the Bio-Rad CFX96 Real-Time System and analyze data using Bio-Rad CFX Maestro Software.

• 42.Prepare the RT-qPCR master mix on ice for Zfp750 and Hprt (housekeeping gene) quantitation using volumes as listed below.

RT-qPCR master mix

Reagent	Amount per sample
10 μM Forward primer	0.5 μL
10 μM Reverse primer	0.5 μL
2× Maxima SYBR Green qPCR Master Mix	7 μL
Nuclease-free water	5 μL
Total	13 μL

Prepare fresh or store at 4°C for less than 1 day.

• 43.
  Prepare RT-qPCR reaction for the control epidermis.

  • a.
    Add 1 μL of cDNA derived from the control epidermis to the bottom of two wells in a 96-well PCR plate.
  • b.
    Add 13 μL of the Zfp750 RT-qPCR master mix to the first well.
  • c.
    Add 13 μL of the Hprt RT-qPCR master mix to the second well.
• 44.
  Prepare RT-qPCR reaction for the Zfp750^fl/fl K14-CreER^tam epidermis.

  • a.
    Add 1 μL of cDNA derived from the epidermis of Zfp750^fl/fl K14-CreER^tam to the bottom of two other wells in the same 96-well PCR plate.
  • b.
    Add 13 μL of the Zfp750 RT-qPCR master mix and the Hprt RT-qPCR master mix to these two wells, respectively.
• 45.Seal the 96-well PCR plate with sealing film, then centrifuge at 250 × g for 1 min.
• 46.Run the RT-qPCR as indicated below.

RT-qPCR cycling conditions

Steps	Temperaturea	Time	Cycles
Initial denaturation	95°C	10 min	N/A
Denaturation	95°C	15 s	40 cycles
Annealing	60°C	30 s	N/A
Extension	72°C	45 s	N/A
Melt curve	65°C	30 s	N/A

^a+0.5°C /cycle, Ramp 0.5°C /s

• 47.Analyze raw data and calculate Zfp750 expressions normalized to Hprt.

Skin collection and digestion for flow cytometry analysis

Timing: ∼3 h

24 h after the last treatment of UVB irradiation, collect the skin tissue for flow cytometry to detect the percentage of immune cells in the skin. We also include a control group without UVB exposure to identify which types of immune cells are altered after UVB treatment.

• 48.Sacrifice mice using carbon dioxide inhalation, followed by cervical dislocation, to ensure they do not perceive pain during this process.
• 49.
  Collect skin samples from the dorsal area immediately after euthanization.

  • a.
    Cut off an approximately 2 cm × 2 cm area from mouse dorsal skin using forceps and scissors.
  • b.
    Wash skin tissue in a 100-mm petri dish containing 10 mL of PBS.
  • c.
    Trim the skin tissue with sterile scissors to remove the subcutaneous fat.
• 50.Transfer the skin tissue to a new 100-mm petri dish and then add 2 mL of skin digestion buffer (Figure 4A).
• 51.Cut the skin tissue into small pieces on ice using sterile scissors, then continue cutting until obtaining a paste-like texture for complete dissociation (Figure 4B).

Note: We recommend having at least 2 people perform this step together to minimize digestion time variations between samples.

CRITICAL: The entire mincing process should be performed on ice and always immerse skin tissue in the digestion buffer to prevent it from drying out. The finer the tissue is minced, the more thorough the subsequent digestion process will be, resulting in a higher yield of cells.

• 52.Transfer 2 mL of the minced skin solution into 15 mL conical tubes.
• 53.Add 2 mL of skin digestion buffer into the Petri dish to rinse and collect any remaining tissue. Transfer the rinsed skin into the same 15 mL conical tubes (Figure 4C).
• 54.Incubate minced skin samples on a rotator at 4°C for 1 h and then incubate these samples on a rotator in a 37°C incubator for another 1 h (Figure 4D).

Note: After 2 h of incubation, the tissue fragments/pellets significantly reduce in size, and the skin digestion buffer becomes turbid.

CRITICAL: If a large number of tissue fragments remain at the bottom of the tubes after this step, extend the 37°C incubation for an additional 10–30 min.

• 55.Pass the cell suspension through a 70 μm cell strainer by gravity into a 50 mL conical tube (Figure 4E).
• 56.Rinse the 15 mL conical tube with 10 mL DMEM/high glucose containing 10% FBS and pass the cell suspension through the strainer into the same 50 mL conical tube.

Note: This step can remove subcutaneous fat, hair, and cell clumps.

• 57.Centrifuge cell suspension at 500 × g for 8 min at 4°C, discard the supernatant and keep the cell pellet (Figure 4F).
• 58.Add 2 mL of red blood cell lysis buffer to cell pellet and gently mix by pipetting. Incubate for 2 min at 20°C–25°C.

CRITICAL: Longer than 5 min of red blood cell lysis process affects cell viability. If there are more than 4 samples, we recommend that more than two people handle these samples simultaneously.

• 59.Stop the lysis by adding 8 mL of FACS buffer. Centrifuge at 500 × g for 3 min at 4°C.
• 60.After centrifuging, discard the supernatant, and resuspend the pellet with 350 μL FACS Buffer.
• 61.Transfer the single cell suspensions to 1.5 mL Eppendorf tubes. Keep on ice until staining.

Note: If there are more than 8 skin samples, we recommend using a 96 v-bottom well plate instead of 1.5 mL Eppendorf tubes for next steps.

Figure 4.

Skin digestion for flow cytometry

(A) Add skin digestion buffer to the skin tissue used for flow cytometry.

(B) Cut skin tissue into small pieces on ice.

(C) Transfer a total of 4 mL of minced skin solution into a 15 mL conical tube.

(D) After 2 h of enzymatic incubation, the minced skin solution becomes turbid, with few obvious tissue fragments remaining.

(E) Pass the cell suspension through the 70 μm strainer into a 50 mL conical tube.

(F) Centrifuge skin suspension at 500 × g for 8 min at 4°C to obtain the skin cell pellet.

Flow cytometry staining of immune cells

Timing: 4 h

Stain the cell suspensions with indicated antibodies for flow cytometric analysis of different immune cells in the skin.

• 62.
  Prepare an unstained negative control

  • a.
    Label a 1.5 mL Eppendorf tube as unstained negative control.
  • b.
    Take 40–50 μL of the single-cell suspension from each sample generated by step 61 and add it to this labeled tube.
  • c.
    Centrifuge unstained control at 500 × g for 3 min at 4°C, discard the supernatant.
  • d.
    Add 150 μL of PBS, centrifuge at 500 × g for 3 min at 4°C, discard the supernatant.
  • e.
    Wash unstained control again as in step 62(d).
  • f.
    Resuspend with 150 μL of 1× BD stabilizing fixative buffer and transfer it into a 5 mL round-bottom flow tube with cell strainer. Keep it at 4°C for further analysis.
• 63.
  Prepare the live/dead control

  • a.
    Gently Vortex OneComp eBeads Compensation Beads for at least 20 s.
  • b.
    Add 1 drop of OneComp eBeads, 100 μL of FACS buffer and 1 μL of BV510 anti-mouse IgG2a antibody to a 5 mL round-bottom flow tube labeled “Live/Dead”. Mix it by flicking and incubate on ice for 30 min in the dark.
  • c.
    Centrifuge live/dead control at 500 × g for 3 min at 4°C, discard the supernatant.
  • d.
    Add 150 μL of PBS, centrifuge at 500 × g for 3 min at 4°C, discard the supernatant.
  • e.
    Wash live/dead control again as in step 63(d).
  • f.
    Resuspend with 150 μL of 1× BD stabilizing fixative buffer and keep it at 4°C for further analysis.
• 64.
  Prepare single-stained control with different fluorochrome

  • a.
    Gently Vortex OneComp eBeads Compensation Beads for at least 20 s to ensure proper suspension.
  • b.
    Label 5 mL round-bottom flow tubes for each of nine fluorophores (PerCP-Cy 5.5, PE-Cy7, APC, Alexa Fluor 700, PE-CF594, APC-eFluor 780, PE, BV605, FITC) that will be used. Add 1 drop of OneComp eBeads and 100 μL of FACS buffer to each labeled tube, and then add 1 μL of the relevant antibody (Table 1). Mix briefly by flicking and incubate on ice for 30 min in the dark.
  • c.
    After incubation, centrifuge single-stained controls at 500 × g for 3 min at 4°C, discard the supernatant.
  • d.
    Wash single-stained controls with 150 μL of PBS.
  • e.
    Centrifuge at 500 × g for 3 min at 4°C, discard the supernatant.
  • f.
    Repeat steps (d) and (e).
  • g.
    Resuspend with 150 μL of 1× BD stabilizing fixative buffer. Keep them at 4°C until data collection.
• 65.
  Flow cytometry staining of immune cells in skin tissue

  • a.
    Centrifuge cell suspension samples from step 61 at 500 × g for 3 min at 4°C, discard the supernatant.
  • b.
    Dilute anti-mouse CD16/32 blocking antibody (Fc block) at a dilution of 1:100 with FACS buffer.
  • c.
    Resuspend cell pellet with 100 μL of Fc block solution and incubate at 20°C–25°C for 15 min.
  • d.
    Centrifuge at 500 × g for 3 min at 4°C and remove the supernatant.
  • e.
    Create an antibody mix by dilution of the nine antibodies listed in Table 1 at their recommended dilutions in one tube of FACS buffer. Prepare 100 μL per sample.
  • f.
    Add 100 μL of antibody mix to each cell sample and gently mix it.
  • g.
    Incubate on ice for 30 min in the dark.
  • h.
    After incubation, centrifuge at 500 × g for 3 min at 4°C and remove supernatant.
  • i.
    Resuspend with 150 μL of PBS, centrifuge at 500 × g for 3 min at 4°C and remove supernatant.
  • j.
    Wash cells again as in step 65(i).
• 66.Dilute Viability Dye eFluor 506 at a dilution of 1:800 in PBS.
• 67.Resuspend the cells with 100 μL of Viability Dye eFluor 506 solution and incubate on ice for 25–30 min in the dark.
• 68.Centrifuge at 500 × g for 3 min at 4°C and remove supernatant.
• 69.Wash cells again as in step 65(i) and (j).

Note: Use the above steps for cell surface staining. If intracellular markers need to be detected, follow the instructions for the Cyto-Fast Fix/Perm Kit after step 69.

• 70.Resuspend the cells with 150 μL of 1× BD stabilizing fixative buffer.
• 71.Transfer cell suspension into 5 mL round-bottom flow tube with cell strainer.
• 72.Centrifuge at 500 × g for 30 s at 4°C. Keep samples on ice or at 4°C until data collection.
• 73.
  Before acquiring samples, use the unstained control, live/dead control, and single-stained controls to set appropriate PMT voltage and adjust compensations. Collect data on a BD LSRFortessa flow cytometer and analyze by using FlowJo software.

  • a.
    The gating strategy of immune panel is shown in Figure 5C. Exclude doublets using FSC-H and FSC-A.
  • b.
    Exclude small debris based on FSC-A and SSC-A under the singlet gate.
  • c.
    Gate viability dye eFluor 506 negative cells to exclude dead cells.
  • d.
    Among live cells, neutrophils are recognized as CD11b⁺ Ly6G⁺, macrophages are recognized as CD11b⁺ F4/80⁺, Dendritic cells are recognized as CD11c⁺ MHC II⁺, B cells are recognized as CD45⁺ CD19⁺, CD4⁺ T cells are recognized as CD45⁺ CD4⁺, and CD8⁺ T cells are recognized as CD45⁺ CD8a⁺. Refer to Table 1 for the fluorophore/channel linked to each antibody.

Table 1.

Immune panel

Fluorophore/Channel	Marker	Clone	Final dilution	Lasers (nm)	Filters
PerCP-Cy5.5	Ly6G	1A8	1/100	488	670/LP
PE-Cy7	CD11b	M1/70	1/100	488	780/60; LP735
APC	CD11c	N418	1/100	635	670/30
Alexa Fluor 700	MHC II	M5/114.15.2	1/100	633	730/45; LP690
PE-CF594	F4/80	BM8	1/100	488	610/20; LP600
APC-eFluor 780	CD4	RM4-5	1/100	633	780/60; LP750
PE	CD19	6D5	1/100	488	585/42; LP556
BV605	CD8a	53–6.7	1/100	405	605/40; LP595
FITC	CD45	30-F11	1/100	488	530/30; LP502

Figure 5.

Zfp750 gene deletion in mouse epidermis and UVB-induced skin inflammation

(A) RT-qPCR of Zfp750 mRNA expression in the control and Zfp750^−/− epidermis. Normalize qPCR result to Hprt. n = 3, mean values are shown with error bars representing SEM. ∗∗∗p < 0.001 (t test).

(B) Images of mouse dorsal skin during UVB treatment. Depilate the mice and administer the first dose of UVB on day 1, the second dose on day 2, and harvest them 24 h after the last UVB treatment on day 3.

(C) Gating strategy for neutrophils in the skin cell suspension with or without UVB radiation.

Expected outcomes

The epidermis can be easily peeled off after Dispase digestion, yielding over 3 μg of total RNA from the isolated epidermis, with RNA concentrations above 50 ng/μL. As shown in Figure 5A, RT-qPCR result shows a significant reduction in Zfp750 gene expression in the knockout mice epidermis compared to the control, confirming the success of tamoxifen-induced epidermal gene ablation.

In Figure 5B, following hair removal and initial UVB irradiation on Day 1, no obvious skin inflammation or damage is visible. On Day 2, 24 h after the first UVB exposure, mouse skin shows very mild redness. While on Day 3, 24 h after the second UVB exposure, mice exhibit skin inflammatory responses, such as erythema. Flow cytometry results show that only about 0.1–0.8% of the cells were neutrophils in the unirradiated mouse skin. 24 h after the first UVB irradiation, 3–5% of the cells in the mouse skin are neutrophils, and after 24 h following the second UVB irradiation, the proportion of neutrophils increase to 7–10% (Table 2). This indicates that UVB-induced inflammation can increase neutrophil recruitment in the skin³ (Figure 5C).

Table 2.

Total collected events by flow cytometer and the percentage of expected populations

Immune panel	Without UVB treatment	1st UVB irradiation	2nd UVB irradiation
Collected events	200,000–400,000	200,000–400,000	200,000–400,000
Live cells	50%–70%	50%–70%	50%–70%
Neutrophils	0.1%–0.8%	3%–5%	7%–10%

Limitations

Nair treatment may lead to skin irritation in mice before UVB treatment which could influence final experimental results. To minimize unexpected damage to skin, we recommend reducing the duration of Nair application and removing the Nair lotion thoroughly and gently using wet wipes.

The immune panel used in this protocol is for basic analysis of major immune cells in unirradiated or UVB-irradiated mouse whole skin. Although epidermis and dermis work together to establish the skin defense system against invading pathogens,⁴ it is necessary to separate the epidermis from dermis and perform flow cytometry on each layer individually to further distinguish their roles during UVB-irradiated skin inflammation.⁵

Troubleshooting

Problem 1

Difficult to separate the epidermis from the dermis (Related to epidermal isolation for gene expression analysis section).

Potential solution

Ensure that the subcutaneous fat layer is thoroughly removed, as any residual fat tissue can affect Dispase penetration into the skin, thereby reducing the efficacy of enzymatic digestion. Additionally, after the digestion is complete, use delicate task wipers to absorb moisture from the edges of the skin. Then, when placing the skin into a new 60 mm dish, the dry epidermis should easily adhere to the bottom of the dish. After incubation with Dispase, if the epidermis is still difficult to separate, the incubation time can be extended to 18–20 h, and gently scrape off epidermis using a scalpel.

Problem 2

Low RNA yield of mouse epidermis (Related to epidermal RNA extraction and cDNA synthesis section).

Potential solution

Use a scalpel to chop the epidermis into small pieces as much as possible. After adding the RLT Plus lysis buffer, thoroughly pipette the mixture until no obvious epidermal tissue is visible.

Before passing the RNA lysate through the gDNA Eliminator spin column, be sure to centrifuge at 12,000 × g for 5 min to remove any precipitate. When transferring the supernatant, try to avoid touching the bottom precipitate to prevent clogging the column.

Problem 3

Gene is not deleted in mouse epidermis (Related to steps 1–5).

Potential solution

Ensure the concentration of tamoxifen solution is correct.

Ensure that tamoxifen is not expired and has not experienced multiple freeze-thaw cycles.

Vigorously vortex tamoxifen solution to fully dissolve any crystals before i.p. injection.

After the injection, check if the injection site has swelling, bleeding, or other abnormalities. A successful i.p. injection does not result in any noticeable lumps at the injection site.

Problem 4

UVB-induced skin inflammation does not develop (Related to expose depilated mice to UVB radiation section).

Potential solution

Make sure the mice used for this protocol are in telogen phase. Pink or flesh-colored skin indicates the telogen phase while darker or blackish skin indicates the anagen phase with active growth of hair shafts.²

Ensure the UVB irradiance is measured accurately and calculate the UVB exposure time using the formula provided in Figure 2E.

Problem 5

Small cell pellet after skin digestion (Related to skin collection and digestion for flow cytometry analysis section).

Potential solution

Ensure the concentrations of Liberase, Collagenase D and DNase I are correct.

Cutting the skin into paste-like pieces until fully dissociated and maintaining rotation during the incubation procedure can enhance thorough enzymatic digestion.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, George L. Sen (gsen@health.ucsd.edu).

Technical contact

Further information and requests for technical advice should be directed to and will be fulfilled by the technical contact, Ye Liu (yel024@health.ucsd.edu).

Materials availability

This study did not generate any unique materials.

Data and code availability

The study did not generate any unique datasets or code.

Acknowledgments

This work was supported by grants from the National Institutes of Health (NIH R01AR081215 and R01AR066530) to G.L.S.

Author contributions

Conceptualization, Y.L. and G.L.S.; investigation, Y.L. and G.L.S.; writing, Y.L. and G.L.S.; supervision, G.L.S.

Declaration of interests

The authors declare no competing interests.